WO2024185847A1 - Genetically modified mouse - Google Patents

Abstract

The present invention provides novel technology which makes it possible to control the function of a protein in the living body of mouse with high temporal resolution.　Provided is a genetically modified mouse, said genetically modified mouse comprising: a pair of Cre recombinase recognition sequences; a transcription termination polyA sequence that is located between the pair of Cre recombinase recognition sequences; a transport inhibitor response 1 (TIR1) family gene sequence that is located downstream of the pair of Cre recombinase recognition sequences and that codes for a TIR1 family protein; and a promoter sequence that is located upstream of the pair of Cre recombinase recognition sequences, and that can drive expression of the TIR1 family gene after recombination of the pair of Cre recombinase recognition sequences.

Description

Genetically engineered mice

The present invention relates to genetically modified mice and methods for inducing the degradation of target proteins.

Technologies that selectively inhibit or destroy the function of specific genes in vivo as a method for analyzing protein functions have made a major contribution to life science research. A representative example is the technology for producing knockout mice, which is based on gene targeting of the mouse ES cell genome using homologous recombination, and has made great progress in elucidating gene functions in vivo and the mechanisms of disease onset.

　Subsequently, conditional knockout technology was developed, making it possible to knock out genes in specific tissues or cell types. In conditional knockout technology, mice (called flox mice) are created in which a pair of loxP sequences recognized by Cre recombinase is introduced before and after the target gene region, and the target gene is destroyed in a tissue- or cell-type-specific manner by mating this flox mouse with a mouse that expresses Cre recombinase in a tissue- or cell-type-specific manner. Conditional knockout technology has made it possible to study gene functions in adults even for genes that are lethal during development when knocked out throughout the body.

In conditional knockout technology, CreER recombinase, which has the property of inducing recombination activity by drug administration, is used as the Cre recombinase, making it possible to perform tissue- or cell-type-specific and time-specific gene destruction.

However, conditional knockout technology is still unable to transiently suppress the function of the protein encoded by the manipulated gene with high temporal resolution. For example, when using CreER recombinase, which can induce recombination activity by drug administration, even after gene destruction is induced by drug administration, the protein expressed from the gene before destruction remains for a certain period of time, so it takes a long time for the expression of the target protein to actually decrease and its activity to disappear.

The above problems are a major issue in drug discovery when selecting target proteins before developing a therapeutic drug. The impact on biological functions when inhibiting the function of a candidate protein over the long term by destroying the candidate gene using whole-body knockout or conditional knockout does not necessarily match the impact when the protein's function is inhibited temporarily by administering the actual therapeutic drug to an animal, and sometimes unexpected side effects become apparent only after the actual therapeutic drug is administered. In such cases, the enormous amount of effort and investment required to develop the therapeutic drug is wasted, which is a major problem.

International Publication No. 2021/009993

The objective of the present invention is to provide a new technology that enables tissue- or cell-type-specific control of protein function in vivo with high temporal resolution.

The auxin degron method is a technology that involves introducing the Transport Inhibitor Response 1 (TIR1) family protein, which constitutes an auxin-responsive ubiquitin ligase, into cells and controlling the degradation of target proteins fused with a degradation tag (called a degron) by adding the plant hormone auxin or an auxin analogue.

In order to achieve the above object, the present inventors have generated Rosa26 OsTIR1(F74G) mice by introducing a construct in which a LoxP-STOP-LoxP (LSL) sequence is arranged upstream of a cDNA sequence encoding a rice-derived TIR1 protein having an F74G mutation (hereinafter referred to as "OsTIR1(F74G) protein") into the Rosa26 locus. The Rosa26 OsTIR1 ^{( F74G)} mice were crossed with transgenic (Tg) mice expressing Cre recombinase in a specific tissue or cell type-specific manner to generate mice expressing OsTIR1(F74G) protein in a tissue or cell type-specific manner. The present inventors have found that administration of an auxin analog to the mice induced transient degradation of a degron-fused target protein very quickly, and the original expression level was then rapidly restored. Furthermore, administration of an auxin analogue to pregnant mice induced protein degradation in fetal mice, and administration of an auxin analogue to lactating female mice induced protein degradation in newborn mice, demonstrating that degradation of membrane proteins as target proteins can also be induced in vivo in mice.

The present invention is based on the above research results and provides the following.
(1) A genetically modified mouse,
A pair of Cre recombinase recognition sequences,
A transcription termination polyA sequence located between the pair of Cre recombinase recognition sequences;
The genetically modified mouse comprises: a Transport Inhibitor Response 1 (TIR1) family gene sequence encoding a TIR1 family protein, which is located downstream of the pair of Cre recombinase recognition sequences; and a promoter sequence, which is located upstream of the pair of Cre recombinase recognition sequences and is capable of driving expression of the TIR1 family gene after recombination between the pair of Cre recombinase recognition sequences.
(2) The genetically modified mouse described in (1), in which in the TIR1 family protein, the Phe residue corresponding to position 74 of the amino acid sequence shown in SEQ ID NO: 1 is replaced with a Gly residue, an Ala residue, or a Ser residue.
(3) A genetically modified mouse described in (1) or (2), which contains the pair of Cre recombinase recognition sequences, the transcription termination polyA sequence, the TIR1 family gene sequence, and the promoter sequence in the Rosa26 locus.
(4) The genetically modified mouse according to any one of (1) to (3), further comprising a gene encoding a target protein to which a degron sequence has been added and/or a gene encoding Cre recombinase.
(5) The genetically modified mouse described in (4), wherein the gene encoding the target protein is essential for development and/or survival.
(6) The genetically modified mouse according to (4) or (5), wherein the target protein is a membrane protein.
(7) The genetically modified mouse according to any one of (4) to (6), wherein the gene encoding the Cre recombinase is placed under the control of a site-specific promoter.
(8) The genetically modified mouse described in (7), wherein the function of the gene encoding the target protein at the site where the site-specific promoter drives expression is essential for development and/or survival.
(9) A sperm, a fertilized egg, or an embryo generated in a genetically modified mouse according to any one of (1) to (8).
(10) A cell derived from a genetically modified mouse according to any one of (1) to (8).
(11) A method for inducing degradation of a target protein in a mouse body, comprising:
A degradation induction step of administering an auxin or an auxin analog to a genetically modified mouse to induce degradation of a target protein to which a degron sequence has been added,
The genetically modified mouse comprises a TIR1 family gene expression system, a gene encoding a target protein to which the degron sequence has been added, and a gene encoding a Cre recombinase;
The TIR1 family gene expression system comprises:
A pair of Cre recombinase recognition sequences,
A transcription termination polyA sequence located between the pair of Cre recombinase recognition sequences;
the method comprising: a TIR1 family gene sequence encoding a Transport Inhibitor Response 1 (TIR1) family protein, located downstream of the pair of Cre recombinase recognition sequences; and a promoter sequence located upstream of the pair of Cre recombinase recognition sequences, which is substantially incapable of driving expression of the TIR1 family gene before recombination between the pair of Cre recombinase recognition sequences, but is capable of driving expression of the TIR1 family gene after recombination between the pair of Cre recombinase recognition sequences.
(12) The method according to (11), wherein the amount of the auxin or auxin analog administered is 0.1 mg/kg body weight to 50 mg/kg body weight.
(13) The method according to (11) or (12), further comprising an evaluation step of evaluating the phenotype of the genetically modified mouse after the degradation induction step.
(14) The method according to (13), wherein the evaluation step is carried out at least 3 hours after the decomposition induction step.
(15) The method according to any one of (11) to (14), wherein the genetically modified mouse is a fetus or a preweaned mouse, and an auxin or an auxin analog is administered to the mother mouse in the degradation induction step.
(16) The method according to any one of (11) to (15), wherein the genetically modified mouse is a disease model mouse, and the target protein is a candidate protein for drug discovery target.
(17) The method according to (16), wherein disruption based on whole-body knockout or conditional knockout of a gene encoding the candidate drug discovery target protein has no therapeutic or preventive effect on the disease model mouse or exhibits (harmful) side effects.
(18) The method further comprises administering to the genetically modified mouse a drug targeting a factor other than the drug discovery target candidate protein;
The method according to (16) or (17), wherein the evaluation step comprises comparing a phenotype obtained when both the candidate drug discovery target protein and the factor are targeted with a phenotype obtained when only the factor is targeted.
(19) A method for inducing degradation of a target protein in a mouse-derived cell, comprising the steps of:
(10) A culturing step of culturing the cell according to (10);
The method described above, further comprising a degradation inducing step of adding an auxin or an auxin analog to the cells to induce degradation of the target protein to which the degron sequence has been added.
This specification includes the disclosure of Japanese Patent Application No. 2023-036211, which is the priority basis of this application.

The present invention provides a genetically modified mouse that allows for tissue- or cell-type-specific control of protein function with high temporal resolution in vivo.

A diagram showing the method for generating the Rosa26 ^OsTIR1(F74G) mouse line. Figure 1A shows homologous recombination at the Rosa26 locus using a Target Vector containing a CAG promoter (pCAG), loxP-neor-STOP-loxP sequence, OsTIR1(F74G) cDNA sequence, and ires-EGFP sequence. Figure 1B shows the generated Rosa26 ^OsTIR1(F74G) allele. FIG. 13 shows that the loxP-neor-STOP-loxP sequence is removed by site-specific recombination of the loxP sequence by Cre recombinase, thereby inducing expression of the OsTIR1(F74G) protein. This is a diagram showing a time series analysis of protein degradation in a mouse in vivo. FIG. 3A shows the crossbreeding for producing Rosa26 ^OsTIR1/+ :Satb1 ^Venus-mAID/+ :Cd4Cre mice. FIG. 3B shows the results of flow cytometry analysis of the fluorescence intensity of Satb1-Venus in peripheral blood T cells after administration of 5-ph-IAA at a dose of 0.1 mg/body. FIG. 3C shows the results of flow cytometry analysis of the fluorescence intensity of Satb1-Venus in peripheral blood T cells after administration of 5-ph-IAA at a dose of 0.02 mg/body. FIG. 4 is a graph showing the results of FIG. 3B and FIG. 3C. 5-ph-IAA was administered to pregnant or lactating female mice, and the fluorescence intensity of Satb1-Venus in T cells in the thymus excised from fetal or neonatal mice was analyzed by flow cytometry. This figure shows the results of inducing protein degradation in vitro by targeting the membrane protein PD-1. Figure 7 shows the results of inducing protein degradation in vivo in mice by targeting the membrane protein PD-1. Figure 7A shows the crossbreeding for generating Rosa26 ^OsTIR1/+ :Pdcd1-mAID:Vav1Cre mice. Figure 7B shows the results of flow cytometry analysis of PD-1 protein expression in follicular T cells in Peyer's patches 12 hours after intraperitoneal administration of 5-ph-IAA or PBS. FIG. 8 shows the results of inducing PD1 degradation in PD1 degron mice (Rosa-OsTIR1/+; Pdcd1 ^mAID/mAID ; Vavcre) expressing OsTIR1 in whole blood cells transplanted with MC38 colon cancer. FIG. 8A shows an outline of the experiment. FIG. 8B shows the results of measuring the expression level of PD1 in tumor-infiltrating cells (TIL). FIG. 8C shows the results of measuring the tumor diameter in the PBS-administered group and the 5-ph-IAA-administered group. FIG. 9A shows the results of inducing PD1 degradation in PD1 degron mice (Rosa-OsTIR1/+; Pdcd1 ^mAID/mAID ; E8Icre mice) expressing OsTIR1 only in CD8 T cells transplanted with MC38 colon cancer. FIG. 9A shows the results of measuring the tumor diameter in the PBS-administered group and the 5-ph-IAA-administered group of the PD1 degron mice. FIG. 9B shows the results of comparing the tumor diameter when 5-ph-IAA was administered to PD1 degron mice expressing OsTIR1 only in CD8 T cells (Rosa-OsTIR1/+; Pdcd1 ^mAID/mAID ; E8Icre mice) and PD1 degron mice expressing OsTIR1 in all blood cells (Rosa-OsTIR1/+; Pdcd1 ^mAID/mAID ; Vavcre).

1. Genetically Modified Mouse 1-1. Overview The first aspect of the present invention is a genetically modified mouse. The genetically modified mouse of this aspect includes a gene expression system capable of inducing expression of a TIR1 family gene after recombination between a pair of Cre recombinase recognition sequences. In the genetically modified mouse of this aspect, expression of a TIR1 family protein is induced by tissue- or cell-type-specific expression of Cre recombinase, and further, degradation of a target protein to which a degron sequence has been added can be transiently induced by administration of auxin or an auxin analog at any time point.

1-2. Definitions The following terms frequently used in this specification are defined below.
In the present specification, the mouse strain or genetic background of the "genetically modified mouse (transgenic mouse)" is not limited, and may be, for example, a C57BL/6N strain, a C57BL/6J strain, a BALB/C strain, a 129 strain, or the like, or may be a hybrid strain obtained by crossing a plurality of strains. The genetically modified mouse may be, for example, a knock-in (KI) mouse, or a transgenic mouse other than a KI mouse (e.g., a Tg mouse in which a foreign gene sequence is introduced at a random position on the genome). The method of producing the genetically modified mouse is not limited, and may be, for example, a genetically modified mouse produced by gene targeting based on conventional homologous recombination or a genome editing method using the CRISPR/Cas9 system. The genetically modified mouse may be at any developmental stage or week/fetal age, and may be, for example, a fetal mouse, a perinatal mouse, a neonatal mouse, a preweaning mouse, a young adult mouse, or an adult mouse. Furthermore, the genetically modified mouse may be either male or female. In addition, the genetically modified mouse may be a disease model mouse, for example, a mouse that has developed a disease or may develop a disease due to genetic mutation, genetic abnormality, gene introduction, drug treatment, and/or surgical intervention, etc.

In this specification, the term "TIR1 (Transport Inhibitor Response 1) family protein" refers to an F-box protein, which is one of the subunits that form the E3 ubiquitination complex (SCF complex) in the protein degradation of the ubiquitin/proteasome system. Plant-derived TIR1 family proteins function as receptors for the growth hormone auxin, and are known to recognize the Aux/IAA family proteins, which are inhibitors of the auxin signaling system, and degrade target proteins depending on the binding with auxin. In this specification, the plant species from which the TIR1 family proteins are derived is not limited, and may be, for example, Arabidopsis thaliana, rice, zinnia, pine, fern, Physcomitrella patens, etc. A specific example is the rice-derived TIR1 protein (hereinafter referred to as "OsTIR1 protein"), which consists of the amino acid sequence shown in SEQ ID NO: 1.

As used herein, "TIR1 family gene sequence" refers to the base sequence or nucleic acid sequence of a gene encoding a TIR1 family protein. There is no limitation on the type of TIR1 family gene sequence, so long as it is a gene encoding a plant-derived TIR1 family protein. There is also no limitation on the type of plant from which it is derived, and it may be derived, for example, from Arabidopsis thaliana, rice, zinnia, pine, fern, Physcomitrella patens, etc. Specific examples of TIR1 family gene sequences include the base sequences of the TIR1 gene, AFB1 gene, AFB2 gene, AFB3 gene, FBX14 gene, and AFB5 gene. Among them, the OsTIR1 gene, which is a TIR1 family gene derived from rice, is preferred, and examples of such genes include the genes with NCBI accession numbers NM_001059194 (GeneID: 4335696), Os04g0395600, or accession numbers EAY93933 and OsI_15707, and more specifically, the OsTIR1 gene sequence consisting of the nucleotide sequence shown in SEQ ID NO: 2, which encodes the OsTIR1 protein consisting of the amino acid sequence shown in SEQ ID NO: 1. The TIR1 family gene sequences such as the OsTIR1 gene sequence may also be codon-optimized for mouse cells, etc.

In this specification, the term "auxin degron system" refers to a protein degradation control system that applies the plant protein degradation system based on the plant hormone auxin to host cells such as eukaryotic cells of non-plant origin. Specifically, this system is a system in which the above-mentioned TIR1 family protein guides a target protein labeled with a degron to degradation using the ubiquitin/proteasome degradation system of the host cell in a manner dependent on binding with auxin or an auxin analog.

In this specification, "auxin" refers to a group of plant hormones that control plant growth. In this specification, auxin includes both naturally occurring auxins and non-natural auxins (synthetic auxins) that exhibit physiological actions similar to those of auxins. Known naturally occurring auxins include indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4Cl-IAA). Known synthetic auxins include naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The structure of indole-3-acetic acid (IAA), one of the natural auxins, is shown in the following formula (I).

In this specification, the term "auxin analog" is not particularly limited as long as it has a structure similar to any of the above-mentioned auxins, such as natural auxins such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4Cl-IAA), and can bind to a TIR1 family protein to induce degradation of a target protein labeled with a degron. Examples of auxin analogs are disclosed in WO 2021/009993, and may be, for example, a compound represented by the following general formula (II) or an ester thereof, or a compound represented by the following general formula (III).

［式中、Ｒ^１は、置換基を有していてもよく、環を構成する炭素原子の一部がヘテロ原子で置換されていてもよい環状の脂肪族炭化水素基、又は、置換基を有していてもよく、環を構成する炭素原子の一部がヘテロ原子で置換されていてもよい芳香族炭化水素基である。］

[In the formula, R ¹ is a cyclic aliphatic hydrocarbon group which may have a substituent and in which a portion of the carbon atoms constituting the ring may be substituted with a heteroatom, or an aromatic hydrocarbon group which may have a substituent and in which a portion of the carbon atoms constituting the ring may be substituted with a heteroatom.]

［式中、Ｒ^２は、水素原子、炭素数１～６のアルキル基、又はハロゲン原子を表し、Ｒ^３は、水素原子、又は炭素数１～６のアルキル基を表す。但し、Ｒ^２が、水素原子、又は炭素数１～６のアルキル基である場合、Ｒ^３は、炭素数１～６のアルキル基である。］

[In the formula, R ² represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom, and R ³ represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. However, when R ² is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, R ³ is an alkyl group having 1 to 6 carbon atoms.]

Another specific example of an auxin analog is 5-phenyl-indole-3-acetic acid (hereinafter referred to as "5-ph-IAA"), shown in the following formula (IV).

In this specification, "multiple" refers to an integer of 2 or more, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3. In addition, in this specification, "several" refers to, for example, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

As used herein, "amino acid identity (amino acid sequence identity)" refers to the percentage of matching amino acid residues out of the total number of amino acid residues in the amino acid sequences of two polypeptides being compared, when aligned by inserting appropriate gaps into one or both, as necessary, to maximize the number of matching amino acid residues. "Base identity (base sequence identity)" can also be determined in a similar manner.

As used herein, "amino acid substitution" refers to substitutions between the 20 types of amino acids that make up natural proteins. Amino acid substitutions are preferably within conservative amino acid groups that have similar properties such as charge, side chain, polarity, and aromaticity. Examples include substitutions within the uncharged polar amino acids with low polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp).

1-3. Configuration The genetically modified mouse of the present invention comprises a pair of Cre recombinase recognition sequences, a transcription termination polyA sequence located between the pair of Cre recombinase recognition sequences, a TIR1 family gene sequence located downstream of the pair of Cre recombinase recognition sequences, and a promoter sequence located upstream of the pair of Cre recombinase recognition sequences.

In this specification, the entire sequence region comprising a pair of Cre recombinase recognition sequences, a transcription termination polyA sequence located between the pair of Cre recombinase recognition sequences, a TIR1 family gene sequence located downstream of the pair of Cre recombinase recognition sequences, and a promoter sequence located upstream of the pair of Cre recombinase recognition sequences, which is capable of expressing a TIR1 family gene after recombination between the pair of Cre recombinase recognition sequences, and which is composed of the TIR1 gene and an expression control region necessary for the gene expression, is referred to as a "TIR1 gene expression system" or simply a "gene expression system." Thus, the genetically modified mouse of the present invention comprises a TIR1 gene expression system.

In the TIR1 gene expression system of the genetically modified mouse of the present invention, the promoter sequence is located upstream of a pair of Cre recombinase recognition sequences, and the TIR1 family gene sequence is located downstream of a pair of Cre recombinase recognition sequences. Here, "upstream" and "downstream" are defined based on the direction of transcription induced from the promoter. In other words, in the transcript transcribed from the promoter, the 5' side corresponds to the upstream side, and the 3' side corresponds to the downstream side.

In the genetically modified mouse of the present invention, the TIR1 gene expression system may be contained in the genome, or a vector such as an expression vector (e.g., a plasmid vector) containing the TIR1 gene expression system may be introduced into the genetically modified mouse. When the TIR1 gene expression system is contained in the genome, it can be introduced into any position in the genome, but it is preferable to introduce it into a gene locus that can be expressed widely regardless of cell type or developmental stage and that is unlikely to affect the expression or function of other endogenous genes, such as the Rosa26 gene locus or the β-actin gene locus.

In one embodiment, the genetically modified mouse comprises a pair of Cre recombinase recognition sequences, a transcription termination polyA sequence, a TIR1 family gene sequence, and a promoter sequence in the Rosa26 locus (i.e., comprises a TIR1 gene expression system in the Rosa26 locus). As used herein, the "Rosa26 locus" is a genomic region on mouse chromosome 6, also referred to as Gt(ROSA)26Sor. The position at which the TIR1 gene expression system is introduced in the Rosa26 locus is not particularly limited. For example, stable expression can be obtained by introducing the TIR1 gene expression system into the intron region between exon 1 and exon 2 of the Rosa26 locus.

In the genetically modified mouse of the present invention, the TIR1 family gene sequence is a nucleotide sequence or nucleic acid sequence encoding any TIR1 family protein. Examples include nucleotide sequences or nucleic acid sequences encoding wild-type or mutant TIR1 family proteins derived from Arabidopsis thaliana, rice, zinnia, pine, fern, or Physcomitrella patens. An example of a nucleotide sequence or nucleic acid sequence encoding a wild-type TIR1 family protein derived from rice is a nucleotide sequence encoding the OsTIR1 protein derived from rice having the amino acid sequence shown in SEQ ID NO:1, for example an OsTIR1 gene sequence having the nucleotide sequence shown in SEQ ID NO:2. Examples of mutant TIR1 family proteins include an amino acid sequence in which one or several (e.g., 1 to 100, 1 to 50, 1 to 20, 1 to 10, or 1 to 5) amino acids have been deleted, inserted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, or a protein consisting of or containing an amino acid sequence having 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 1. This identity of the amino acid sequence can be calculated for the entire length of the amino acid sequence shown in SEQ ID NO: 1, or can be calculated for the amino acid sequence shown in SEQ ID NO: 1 excluding position 74, as described below. It is preferable that the wild-type or mutant TIR1 family protein has an activity equivalent to or greater than that of wild-type TIR1, i.e., the activity of recognizing a degron in the presence of auxin or an auxin analog and leading to the degradation of a target protein.

In one embodiment, in the TIR1 family protein in the genetically modified mouse or TIR1 gene expression system of the present invention, the Phe residue corresponding to position 74 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a Gly residue, an Ala residue, or a Ser residue (referred to as "F74G mutation", "F74A mutation", "F74S mutation", etc. in the present specification). An example of a rice-derived TIR1 protein in which the Phe residue corresponding to position 74 of the amino acid sequence shown in SEQ ID NO:1 is replaced with a Gly residue is an example of the amino acid sequence shown in SEQ ID NO:3. An example of a rice-derived TIR1 protein in which the Phe residue corresponding to position 74 of the amino acid sequence shown in SEQ ID NO:1 is replaced with an Ala residue is an example of the amino acid sequence shown in SEQ ID NO:4. The TIR1 family protein in this embodiment may be one that consists of or contains an amino acid sequence that has 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO:1, except for position 74 of the amino acid sequence shown in SEQ ID NO:1.

As used herein, the term "degron sequence" refers to an amino acid sequence that can induce degradation by the auxin degron system when added to a target protein. For example, a plant-derived Aux/IAA family protein or a fragment thereof functions as a degron sequence and can therefore be used as a degron sequence in the genetically modified mouse or TIR1 gene expression system of the present invention.

The gene encoding the Aux/IAA family protein is not particularly limited in type, so long as it is a plant-derived Aux/IAA family gene. Examples of Arabidopsis-derived Aux/IAA family genes include the IAA1 to IAA34 genes, and preferably the IAA17 gene. The sequences of Arabidopsis-derived Aux/IAA family genes are registered in TAIR (The Arabidopsis Information Resource) and are available as needed. A fragment of a plant-derived Aux/IAA family protein that can be used as a degron sequence preferably has an activity of binding to a TIR1 family protein/auxin or auxin analog complex and leading to the degradation of a target protein, and may be, for example, a full-length or partial sequence of mAID, which will be described later.

In this specification, "mAID (Mini-auxin-inducible degron)" refers to a degron sequence consisting of a partial sequence of Arabidopsis thaliana IAA17, which is one of the Aux/IAA family proteins. mAID can be a sequence consisting of a region containing at least two Lys residues on the N-terminal and C-terminal sides of the domain II region of an Aux/IAA family protein, or a sequence consisting of two or more such sequences linked together. A specific example of an mAID is the amino acid sequence shown in SEQ ID NO:5.

As used herein, the term "Cre recombinase recognition sequence" refers to a sequence that is recognized by Cre recombinase and can induce recombination between two sequences. Specific base sequences of Cre recombinase recognition sequences are known in the art, and examples include loxP sequences (ATAACTTCGTATAGCATACATTATACGAAGTTAT, SEQ ID NO: 6); lox511 sequences (ATAACTTCGTATAGTATACATTATACGAAGTTAT, SEQ ID NO: 7); lox2272 sequences (ATAACTTCGTATAGGATACTTTATACGAAGTTAT, SEQ ID NO: 8); and loxFAS sequences (ATAACTTCGTATATACCTTTCTATACGAAGTTAT, SEQ ID NO: 9). In the genetically modified mouse or TIR1 gene expression system of the present invention, a pair of Cre recombinase recognition sequences is located between the TIR1 family gene sequence and the promoter sequence, and a transcription termination polyA sequence is located between the pair of Cre recombinase recognition sequences. A pair of Cre recombinase recognition sequences are generally arranged in the same direction. When a pair of Cre recombinase recognition sequences are present in the same direction, Cre recombinase recognizes the two sequences and induces site-specific recombination between them, resulting in the excision and removal of the sequence sandwiched between the two sequences.

As used herein, the term "transcription termination polyA sequence" refers to a base sequence capable of causing the termination of transcription. The transcription termination polyA sequence is also called a polyadenylation signal or transcription terminator, and can induce the termination of transcription and the polyadenylation of the 3' end of the transcription product. In the genetically modified mouse or TIR1 gene expression system of the present invention, the transcription termination polyA sequence is arranged between a pair of Cre recombinase recognition sequences, and transcription can be terminated on that sequence. The type of transcription termination polyA sequence is not particularly limited, and any transcription termination polyA sequence can be used. For example, a transcription termination polyA sequence derived from the human growth hormone (HGH) gene, the SV40 gene, the rabbit β-globin gene, the bovine growth hormone (BGH) gene, or the phosphoglycerate kinase (PGK) gene may be used, or any known transcription termination polyA sequence that can function in eukaryotic cells may be used. In the genetically modified mouse or TIR1 gene expression system of the present invention, one or more transcription termination polyA sequences can be arranged between a pair of Cre recombinase recognition sequences. For example, placing two or more transcription termination polyA sequences is advantageous because it is possible to suppress leaky expression of genes placed downstream.

In the genetically modified mouse or TIR1 gene expression system of the present invention, the "promoter sequence" is a promoter that is located upstream of a pair of Cre recombinase recognition sequences and can drive the expression of the TIR1 family gene located downstream thereof after recombination between the pair of Cre recombinase recognition sequences. Before recombination between the pair of Cre recombinase recognition sequences, transcription induced from the promoter sequence is terminated on the above-mentioned transcription termination polyA sequence, so that the TIR1 family gene sequence located downstream thereof is not transcribed or is not substantially transcribed. The type of promoter sequence is not particularly limited as long as it can drive the expression of genes in the target cell type in which the auxin degron system is to function, and may be either an exogenous promoter or an endogenous promoter, a ubiquitous promoter (systemic promoter) or a site-specific promoter, or may be a constitutively active promoter, an expression-inducible promoter, or a time-specific active promoter. The exogenous promoter may be a promoter derived from a mammal such as a mouse, or a promoter derived from a non-mammalian organism, and for example, a CAG promoter, a CMV promoter, an SV40 promoter, an EF1a promoter, or an RSV promoter can be used. The endogenous promoter may be an endogenous promoter of a locus in the genome into which the TIR1 gene expression system is introduced. For example, when the TIR1 gene expression system is introduced into the Rosa26 gene locus, the expression of the TIR1 family gene may be driven by the endogenous promoter of the Rosa26 gene.

In the genetically modified mouse or TIR1 gene expression system of the present invention, the above promoter sequence is preferably operably linked to the TIR1 family gene located downstream thereof after recombination between a pair of Cre recombinase recognition sequences. As used herein, "operably linked" refers to a functional link between the above promoter sequence and a TIR1 family gene sequence that encodes a TIR1 family protein. This functional link refers to a link that allows transcription of the TIR1 family gene sequence after recombination between a pair of Cre recombinase recognition sequences.

In one embodiment, the genetically modified mouse of the present invention further comprises a gene encoding a target protein to which a degron sequence has been added and/or a gene encoding a Cre recombinase.

In the genetically modified mouse of the above embodiment, there is no limit to the type of target protein to which the degron sequence is added. For example, it may be an exogenous protein or an endogenous protein. The target protein may be, for example, a drug discovery target protein such as a disease-causing protein.

In addition, the position at which the degron sequence is added to the target protein is not limited, and may be, for example, the N-terminus and/or C-terminus of the target protein, or may be inserted into the middle of the amino acid sequence of the target protein. In addition, the number of degron sequences added may be one or more. The gene encoding the target protein to which the degron sequence has been added may be an endogenous gene into which a base sequence encoding the degron sequence has been introduced by knock-in or the like.

In a further embodiment, the gene encoding the target protein is essential for development and/or survival. A gene essential for development here is, for example, a gene that causes developmental abnormalities in an individual when the gene is disrupted by whole-body knockout. A gene essential for survival here is, for example, a gene that causes lethality in at least some individuals when the gene is disrupted by whole-body knockout. Such genes were difficult to analyze using conventional whole-body knockout methods, but can be analyzed by transient functional inhibition using the genetically modified mouse of the present invention.

In a further embodiment, the target protein is a membrane protein. The membrane protein may be, for example, an integral membrane protein, a peripheral membrane protein, or a lipid-anchored protein.

In the above-mentioned embodiment, the type of Cre recombinase is not limited, and may be, for example, a Cre recombinase derived from Escherichia coli P1 phage or a modified form thereof. Examples of modified Cre recombinase include tamoxifen-inducible Cre recombinases such as Cre-ERT and Cre-ERT2.

In a further embodiment, the gene encoding the Cre recombinase is placed under the control of a site-specific promoter, such as a tissue-specific or cell type-specific promoter. Various Cre mice are available in the art, and by selecting and mating Cre mice according to the tissue or cell type of interest, it is possible and convenient to introduce the gene encoding the Cre recombinase placed under the control of a site-specific promoter into a genetically modified mouse.

In a further embodiment, the function of the gene encoding the target protein at the site where the expression is driven by the site-specific promoter is essential for development and/or survival. In this embodiment, a gene essential for development is, for example, a gene that causes developmental abnormalities in an individual when the gene is disrupted by conditional knockout based on the expression of Cre recombinase under the control of the same site-specific promoter. In this embodiment, a gene essential for survival is, for example, a gene that causes lethality in at least some individuals when the gene is disrupted by conditional knockout based on the expression of Cre recombinase under the control of the same site-specific promoter. Although such genes were difficult to analyze using conventional conditional knockout, they can be analyzed by transient functional inhibition using the genetically modified mouse of the present invention.

1-4. Effect In the genetically modified mouse of the present invention, recombination between a pair of Cre recombinase recognition sequences is specifically induced in tissues or cell types where the expression of Cre recombinase is induced by a site-specific promoter, and as a result, the transcription termination polyA sequence located between the pair of Cre recombinase recognition sequences is removed from the TIR1 gene expression system, and the expression of the TIR1 family gene located downstream of the Cre recombinase recognition sequence is induced, resulting in tissue- or cell-type-specific expression of the TIR1 family protein. By administering auxin or an auxin analog to this mouse, the TIR1 family protein binds to the auxin or auxin analog, and the target protein labeled with a degron can be degraded using the ubiquitin/proteasome degradation system of the host cell.

On the other hand, in tissues or cell types in which the expression of Cre recombinase is not induced by a site-specific promoter, the transcription termination polyA sequence is not removed and TIR1 family proteins are not substantially expressed, so that even when auxin or an auxin analog is administered, degradation of the target protein is not induced. Furthermore, when auxin or an auxin analog is not administered, degradation of the target protein labeled with a degron is not induced. Therefore, in the genetically modified mouse of the present invention, degradation of the target protein can be induced in a tissue- or cell-type-specific manner with extremely high temporal resolution.

The present invention also provides sperm, fertilized eggs, or embryos that develop in the genetically modified mouse of the present invention. The sperm, fertilized eggs, or embryos may be frozen sperm, frozen fertilized eggs, or frozen embryos, respectively. Also provided are cells derived from the genetically modified mouse of the present invention, for example, mouse cells described below that are derived from the genetically modified mouse of the present invention.

2. Method for inducing degradation of a target protein 2-1. Overview The second aspect of the present invention is a method for degrading a target protein. The method for degrading a target protein of this aspect can induce degradation of a target protein to which a degron sequence has been added by administering auxin or an auxin analog to a genetically modified mouse or adding it to a mouse cell. In the method of this aspect, degradation of a target protein can be induced in a fetus or a mouse pup before weaning by administering auxin or an auxin analog to a mother mouse. Degradation of a candidate protein as a target for drug discovery can also be induced in a disease model mouse.

2-2. Method The target protein degradation method of the present invention can be carried out on a genetically modified mouse (mouse individual) or mouse cells derived from a genetically modified mouse. The specific configuration of the target protein degradation method of the present invention differs depending on whether the target is a mouse individual or mouse cells. When a genetically modified mouse is used as a target, the target protein degradation method of the present invention includes, as an essential step, a degradation induction step in which auxin or an auxin analog is administered to the genetically modified mouse to induce degradation of a target protein to which a degron sequence has been added, and includes, as a selection step, an evaluation step in which the phenotype of the genetically modified mouse after the degradation induction step is evaluated. When a mouse cell is used as a target, the target protein degradation method of the present invention includes, as an essential step, a culture step in which mouse cells are cultured, and a degradation induction step in which auxin or an auxin analog is added to the mouse cell to induce degradation of a target protein to which a degron sequence has been added, and includes, as a selection step, an evaluation step in which the phenotype of the mouse cell after the degradation induction step is evaluated.

(Decomposition induction process)
In the target protein degradation method of the present invention, the "degradation inducing step" refers, when a genetically modified mouse is the target, to a step of administering auxin or an auxin analogue to the genetically modified mouse to induce degradation of the target protein to which a degron sequence has been added, and, when a mouse cell is the target, to a step of adding auxin or an auxin analogue to the mouse cell to induce degradation of the target protein to which a degron sequence has been added.

In this embodiment, the genetically modified mouse or mouse cell comprises a TIR1 family gene expression system, a gene encoding a target protein to which the degron sequence has been added, and a gene encoding a Cre recombinase. Here, the TIR1 family gene expression system comprises a pair of Cre recombinase recognition sequences, a transcription termination polyA sequence located between the pair of Cre recombinase recognition sequences, a TIR1 family gene sequence encoding a Transport Inhibitor Response 1 (TIR1) family protein located downstream of the pair of Cre recombinase recognition sequences, and a promoter sequence located upstream of the pair of Cre recombinase recognition sequences and capable of driving expression of the TIR1 family gene after recombination between the pair of Cre recombinase recognition sequences. The configurations of the pair of Cre recombinase recognition sequences, the transcription termination polyA sequence, the TIR1 family gene sequence, and the promoter sequence are described in detail in the first embodiment, and therefore will not be described here.

In the method for degrading a target protein of the present invention, the developmental stage or week/fetal age of the genetically modified mouse does not matter. For example, the genetically modified mouse may be a fetal mouse, a perinatal mouse, a newborn mouse, a pre-weaning mouse, a young adult mouse, or an adult mouse.

　The method of administration to genetically modified mice is not limited as long as it is capable of delivering auxin or auxin analogues to tissues or cell types that induce degradation of the target protein. Examples include intravenous administration (tail vein injection), intraperitoneal administration, and subcutaneous administration. In the case of fetal mice or preweaned mice, in addition to administering directly to the mouse in which degradation is to be induced, auxin or auxin analogues can be administered indirectly to the mother mouse via the plasma that crosses the placenta or the milk that is fed. For example, in the case of fetal mice, auxin or auxin analogues can be administered to pregnant mice, and in the case of preweaned pups, auxin or auxin analogues can be administered indirectly to fetal mice or pups by administering them to lactating female mice.

The number of times the auxin or auxin analogue is administered to the genetically modified mouse is not limited. For example, it may be a single administration or multiple administrations, for example, once, twice, three times, four times, or more. The multiple administrations may also be periodic administrations, for example repeated administrations every day, every two days, every three days, or once a week. The same applies to the number of times the auxin or auxin analogue is added to the mouse cells.

When a genetically modified mouse is used as the subject, the dosage of the auxin or auxin analogue is not particularly limited as long as it is capable of inducing degradation of the target protein and does not have a deleterious effect on the genetically modified mouse, and may be, for example, 0.001 mg/kg to 10 g/kg body weight, 0.01 mg/kg to 1 g/kg body weight, or 0.1 mg/kg to 50 mg/kg body weight based on the body weight of the genetically modified mouse, and preferably 0.5 mg/kg to 10 mg/kg body weight or 1 mg/kg to 5 mg/kg body weight. The dosage per administration is not particularly limited, and may be, for example, 0.02 mg to 200 g, 0.2 mg to 20 g, or 2 mg to 1 g, and preferably 10 mg to 200 mg or 20 mg to 100 mg. The dosage can be adjusted depending on, for example, the desired degree of degradation; for example, a dosage aimed at a high level of degradation is 5 mg/kg body weight or more or 10 mg/kg body weight or more, and for example, a dosage aimed at a low level of degradation is 2 mg/kg body weight or less or 1 mg/kg body weight or less. The amount of auxin or auxin analogue added to mouse cells is not particularly limited as long as it is capable of inducing degradation of the target protein and does not have a deleterious effect on the mouse cells, and may be, for example, 0.001 mg/L to 10 g/L, 0.01 mg/L to 1 g/L, or 0.1 mg/L to 50 mg/L.

(Evaluation process)
In the target protein degradation method of the present invention, the "evaluation step" refers to a step of evaluating the phenotype of a genetically modified mouse after the degradation induction step, when a genetically modified mouse is the target, and a step of evaluating the phenotype of mouse cells after the degradation induction step, when mouse cells are the target.

In this specification, the "phenotype of a genetically modified mouse" is not particularly limited as long as it is an observable characteristic exhibited by an individual mouse, or an observable characteristic exhibited by an organ, tissue, cell, body fluid, etc. taken from an individual mouse. The individual-level phenotype may be the appearance or shape of the whole body and body parts; weight, body fat percentage, electrocardiogram, muscle strength such as grip strength; or movement such as walking, sensory function, learning, information processing, feeding, excretion, etc. (behavioral phenotype). The phenotype of an organ or tissue is not limited, and may be, for example, a characteristic observable in an organ such as the skin, heart, liver, intestine, or the central nervous system or peripheral nervous system such as the brain, a characteristic observable in the cells of any organ or tissue, or a characteristic observable in a body fluid such as blood or urine (for example, biomolecules such as proteins, lipids, and nucleic acids in a body fluid).

In this specification, the "phenotype of mouse cells" is not limited to any characteristic that can be observed in a cell, whether it is the whole cell, the inside of the cell (e.g., organelles such as the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, and Golgi apparatus), or the cell surface (e.g., cell membrane and cell surface), but may be, for example, the proliferation ability of a cell or the detected amount of a marker; the expression level or activity of a gene (e.g., nucleic acid such as mRNA or miRNA) or a protein; or the amount of an antigen or a metabolite. The type of mouse cells is not limited, and may be somatic cells or germ cells, or may be stem cells, precursor cells, undifferentiated cells, differentiated cells, or established cell lines (e.g., primary culture cells, cancer cells). Examples of differentiated cells include fibroblasts, epithelial cells, hepatocytes, blood cells, immune cells, mesenchymal cells, nerve cells, and muscle cells.

The specific method of evaluating a phenotype is not particularly limited and can be selected according to the type of phenotype to be evaluated. Specific examples of evaluation methods and means include serum biochemistry tests, immunological tests, electrocardiograms, body weight measurements, body fat measurements, organ weight measurements, behavioral observations, morphological observations, Y-mazes, grip strength tests, open field tests, flow cytometry, northern blotting, in situ hybridization, microarrays, RNA seq, PCR such as RT-PCR, ELISA, immunohistochemistry, and western blotting.

The evaluation step can be carried out at any time after the degradation induction step. For example, the evaluation step may be carried out 1 minute to 1 month, 30 minutes to 1 week, 1 hour to 3 days, 2 hours to 1 day, 3 hours to 12 hours, 5 hours to 10 hours, 7 hours to 8 hours after the degradation induction step, and preferably 3 hours or more later. The evaluation step may be carried out during the period in which the target protein is degraded after the degradation induction step, or may be carried out after the target protein has been degraded and the original expression level has been restored. The time point for carrying out the evaluation step may be selected depending on the desired phenotype.

In one embodiment of the target protein degradation method of this aspect, the target protein is a candidate drug discovery target protein. In this specification, the term "candidate drug discovery target protein" refers to a protein that is a candidate when searching for a protein that is a target for drug discovery. Specifically, it is a candidate protein whose degradation is expected to produce a desired effect, such as a therapeutic or preventive effect on a disease or disease model, or a reduction or avoidance of side effects. In this embodiment, the genetically modified mouse may be a disease model mouse. The specific type of the candidate drug discovery target protein is not limited. For example, it may be a protein whose beneficial effect has been overlooked in previous drug discovery because destruction based on whole-body knockout or conditional knockout of the gene encoding the candidate drug discovery target protein did not show a therapeutic or preventive effect on the disease model mouse or showed (harmful) side effects (e.g., lethality).

In this specification, the term "disease" is not limited, and may be, for example, cancer, hepatitis, infectious disease, etc. Furthermore, the type of "cancer" is not limited, and examples include adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma. Specific types of cancer include, for example, malignant melanoma, thyroid cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, colon cancer, small intestine cancer, prostate cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, biliary tract cancer, brain tumor, head and neck cancer, mesothelioma, osteosarcoma, blood cancer, lymphoma, myeloma, etc.

In a further embodiment, the target protein degradation method of this aspect further includes a step of administering to the genetically modified mouse a drug targeting a factor other than the candidate drug target protein. In this embodiment, the "factor other than the candidate drug target protein" is a factor that is expected to enhance the desired effect, such as a therapeutic or preventive effect on a disease or disease model, or a reduction or avoidance of side effects, by targeting it in combination with the candidate drug target protein, and is, for example, any biological substance such as a protein or nucleic acid other than the candidate drug target protein. The drug targeting a factor other than the candidate drug target protein is not limited and may be a low molecular weight compound, an antibody drug, or a nucleic acid drug such as siRNA or antisense nucleic acid, and may be administered simultaneously with or without the administration of auxin or auxin analog. In this embodiment, the evaluation step can compare the phenotype when both the candidate drug target protein and the factor are targeted with the phenotype when only the factor is targeted. This comparison makes it possible to determine the degree and presence of an effect of enhancing the desired effect, such as a therapeutic or preventive effect against a disease or a reduction or avoidance of side effects, by inducing the degradation of a candidate drug target protein in combination with the administration of a drug that targets a factor different from the candidate drug target protein.

2-3. Effects The method for degrading a target protein of the present invention can control degradation of a target protein in a living mouse body or in mouse cells with extremely high time resolution, thereby achieving transient degradation and subsequent rapid recovery.

In conventional whole-body knockout and conditional knockout, the function of the candidate protein is inhibited for a long period of time by destroying the candidate gene. The phenotype of the mouse in this case may not necessarily match the phenotype when the developed therapeutic drug is actually administered to the animal to transiently control the function of the protein, and unexpected side effects may only become apparent after the developed therapeutic drug is actually administered. In such cases, the enormous investment of labor and cost required for therapeutic development is wasted. In contrast, the targeted protein degradation method of the present invention makes it possible to observe the effects of transiently controlling the function of a protein, making it possible to more accurately verify in advance the actual effects of the therapeutic drug to be developed in the process of searching for candidate proteins for drug discovery, reducing unnecessary investment costs and streamlining the initial stages of drug discovery.

It is also entirely conceivable that there may be effects that cannot be seen with long-term expression suppression using whole-body knockout or conditional knockout (due to the induction of compensatory mechanisms, etc.), but that can only be seen through transient degradation control. Therefore, by targeting candidate proteins for which no therapeutic or other effects could be obtained using conventional whole-body knockout or conditional knockout with the method of the present invention, it is also possible to identify useful candidate proteins that have been overlooked in the early stages of drug discovery.

Example 1: Establishment of Rosa26 ^OsTIR1(F74G) mouse line
(the purpose)
We will generate the Rosa26 ^{OsTIR1(F74G) mouse line, which allows cell type-specific expression of the OsTIR1(F74G} ) protein.

(Methods and Results)
We constructed a target vector in which the cDNA sequence (SEQ ID NO: 10) encoding the OsTIR1(F74G) protein, a rice-derived Transport Inhibitor Response 1 (TIR1) family protein in which the Phe residue at position 74 in the amino acid sequence of the OsTIR1 protein (SEQ ID NO: 1) was replaced with a Gly residue, and the ires-EGFP sequence was placed downstream of a CAG promoter (pCAG) and a loxP-neor-STOP-loxP sequence (SEQ ID NO: 11) (Fig. 1A). We introduced this target vector into ES cells and identified ES clones that had undergone homologous recombination in the intron region between Exon 1 and Exon 2 of the Rosa26 gene (Fig. 1B). We generated chimeric mice from these ES clones, and by obtaining F1 founder mice carrying the desired Rosa26 ^OsTIR1(F74G) allele from these chimeric mice, we established the Rosa26 ^OsTIR1(F74G) mouse line.

Expression of the OsTIR1 ^{(F74G) protein from the Rosa26 OsTIR1} (F74G) allele requires the removal of the loxP-neor-STOP-loxP sequence by site-specific recombination of the loxP sequence using Cre recombinase. By selecting and crossing transgenic (Tg) mice expressing Cre recombinase in a tissue- or cell-specific manner, OsTIR1(F74G) protein can be expressed in a tissue- or cell-specific manner (Fig. 2).

Example 2: Time series analysis of protein degradation in vivo in mice
(the purpose)
Mice expressing a target protein fused with a mini AID degron sequence (mAID) are generated, and these mice are crossed with the Rosa26 ^OsTIR1(F74G) mice generated in Example 1 and Tg mice expressing Cre recombinase. An auxin analog is administered to the mice obtained by crossing to induce degradation of the target protein, and the time course of protein degradation in vivo is analyzed.

(Methods and Results)
By using genome editing to introduce the base sequence encoding the Venus fluorescent protein and the mini AID degron sequence (mAID) into the Satb1 gene, we generated Satb1 ^Venus-mAID mice that express a fusion protein in which the Venus fluorescent protein and the mini AID degron sequence (mAID) are fused to the N-terminus of the Satb1 nuclear protein (Fig. 3A).

Next, the Rosa26 ^OsTIR1(F74G) mice prepared in Example 1, Satb1 ^Venus-mAID mice, and Cd4Cre Tg mice (4-8 week-old male or female; provided by Dr. Chris Wilson, University of Washington) that express Cre recombinase specifically in T cells were crossed to generate Rosa26 ^OsTIR1/+ :Satb1 ^Venus-mAID/+ :Cd4Cre mice (Figure 3A).

5-Phenyl-indole-3-acetic acid (5-ph-IAA) was administered intraperitoneally (ip) to Rosa26 ^OsTIR1/+ :Satb1 ^Venus-mAID/+ :Cd4Cre mice at a dose of 0.1 mg/body or 0.02 mg/body. Blood was collected from the mice at each time point from 0 to 72 hours after administration, and the fluorescence intensity of Satb1-Venus in T cells in the peripheral blood was analyzed by flow cytometry.

The results are shown in Figures 3 and 4. When administered at a dose of 0.1 mg/body, Satb1-Venus expression began to decrease 2-3 hours after administration of 5-ph-IAA, disappeared 6-8 hours later, began to recover 32-48 hours later, and showed almost complete recovery after 72 hours (Figures 3B and 4). When administered at a dose of 0.02 mg/body, Satb1-Venus expression began to decrease 2-3 hours after administration of 5-ph-IAA, showed a significant decrease 5-8 hours later, and recovered after 24 hours (Figures 3C and 4).

These results suggest that administration of 5-ph-IAA to Rosa26 ^OsTIR1/+ :Satb1 ^Venus-mAID/+ :Cd4Cre mice resulted in transient degradation of the target protein in vivo and rapid recovery afterwards. Furthermore, it was shown that the degree of protein degradation could be controlled by adjusting the dose of 5-ph-IAA.

Example 3: Induction of protein degradation in fetal and neonatal mice
(the purpose)
To verify the effect of target protein degradation in fetal and neonatal mice, 5-ph-IAA will be administered to pregnant or lactating female mice, and the degradation of target proteins will be analyzed in fetal or neonatal mice.

(Methods and Results)
5-ph-IAA was intraperitoneally administered at a dose of 0.1 mg/body to pregnant mice (17.5 days of pregnancy) or lactating female mice on the second day after birth for the Rosa26 ^OsTIR1/+ :Satb1 ^Venus-mAID/+ :Cd4Cre mice prepared in Example 2. 24 hours after administration, the thymus was removed from fetal mice or newborn mice lactated by female mice, and the fluorescence intensity of Satb1-Venus in T cells was analyzed by flow cytometry.

The results are shown in Figure 5. Measurement of the fluorescence intensity of Satb1-Venus by flow cytometry revealed that expression of Satb1-Venus was lost in both fetal and neonatal mice. This result demonstrated that administration of 5-ph-IAA to maternal mice can induce protein degradation in fetal and neonatal mice.

Example 4: Targeted degradation of membrane proteins
(the purpose)
The effect of targeting membrane proteins for protein degradation will be verified in cultured cells and in vivo in mice.

(Methods and Results)
(1) Degradation of PD-1 protein in cultured cells We generated T cell lines (Jurkat T cells) expressing a fusion protein in which the mAID degron sequence was fused to the C-terminus of PD-1 protein and OsTIR1(F74G) protein, and added 1 μM 5-ph-IAA or DMSO to the culture medium. 0.5, 1, and 4 hours after addition, the expression levels of PD-1 protein in the T cell lines were analyzed by flow cytometry.

The results are shown in Figure 6. T cell lines treated with 5-ph-IAA showed reduced expression of PD-1 protein compared to control cells treated with DMSO.

(2) Degradation of PD-1 protein in vivo By using genome editing to introduce a base sequence encoding the mini AID degron sequence (mAID) into the PD-1 gene, we generated Pdcd1-mAID mice expressing a fusion protein in which the mini AID degron sequence (mAID) is fused to the C-terminus of the PD-1 protein (Figure 7A).

Next, Rosa26 ^OsTIR1(F74G) mice prepared in Example 1, Pdcd1-mAID mice, and Vav1Cre Tg mice (4-8 week-old male or female; purchased from Jackson Laboratory) that express Cre recombinase specifically in blood cells were crossed to generate Rosa26 ^OsTIR1/+ :Pdcd1-mAID:Vav1Cre mice (Figure 7A).

Rosa26 ^OsTIR1/+ :Pdcd1-mAID: Vav1Cre mice were intraperitoneally administered 5-ph-IAA or PBS at a dose of 0.1 mg/body. Twelve hours after administration, follicular T cells were prepared from Peyer's patches, and PD-1 protein expression was measured by flow cytometry.

The results are shown in Figure 7B. Compared to control mice treated with PBS, PD-1 protein expression was lost in mice treated with 5-ph-IAA.

Example 5: Verification of antitumor effect based on targeted degradation of PD1 protein
(the purpose)
We will transplant colon cancer into the mice created in Example 4, and verify that antitumor immune effects can be obtained by inducing PD1 degradation through administration of 5-ph-IAA. We will also identify the types of blood cells that are important in enhancing antitumor immune responses based on the induction of PD1 degradation.

(Methods and Results)
MC38 colon cancer was transplanted into the PD1 degron mice (Rosa-OsTIR1/+; Pdcd1 ^mAID/mAID ; Vavcre) generated in Example 4. From 10 days after transplantation, PBS or 5-ph-IAA was administered continuously once every two days ( FIG. 8A ), and the tumor diameters in the PBS-administered group and the 5-ph-IAA-administered group were compared.

The results of tumor size measurements are shown in Figure 8C. A significant reduction in tumor size was observed in the 5-ph-IAA administration group. This is similar to the effect of anti-PD1 antibody administration shown in previous literature. In addition, when the expression level of PD1 in tumor-infiltrating cells (TIL) was measured, it was confirmed that PD1 expression was reduced (Figure 8B).

Next, to identify the cell types important for enhancing antitumor immune responses based on PD1 degradation induction, we examined the cell types expressing OsTIR1 using different Cre transgenic mice. Specifically, MC38 colon cancer was transplanted into Rosa-OsTIR1/+;Pdcd1 ^mAID/mAID ;Vavcre mice expressing OsTIR1 in all blood cells, and Rosa-OsTIR1/+;Pdcd1 ^mAID/mAID ;E8Icre mice expressing OsTIR1 only in CD8 T cells, using the same method as above. PBS or 5-ph-IAA was administered continuously, and the tumor diameter was measured.

The results are shown in Figure 9. In Rosa-OsTIR1/+;Pdcd1 ^mAID/mAID ;E8Icre mice expressing OsTIR1 only in CD8 T cells, a significant reduction in tumor size was observed by administration of 5-ph-IAA (Figure 9A). This effect was equivalent to that observed in Rosa-OsTIR1/+;Pdcd1 ^mAID/mAID ;Vavcre mice expressing OsTIR1 in all blood cells (Figure 9B). These results indicate that PD1 degradation only in CD8 T cells is sufficient to enhance antitumor immune responses. This result indicates the advantage of the PD1 degron mouse of the present invention, that the cell type expressing OsTIR1 can be identified by selecting Cre transgenic mice.

These results demonstrate that it is possible to induce the targeted degradation of membrane proteins.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (18)

A genetically modified mouse, comprising:
A pair of Cre recombinase recognition sequences,
A transcription termination polyA sequence located between the pair of Cre recombinase recognition sequences;
The genetically modified mouse comprises: a Transport Inhibitor Response 1 (TIR1) family gene sequence encoding a TIR1 family protein, which is located downstream of the pair of Cre recombinase recognition sequences; and a promoter sequence, which is located upstream of the pair of Cre recombinase recognition sequences and is capable of driving expression of the TIR1 family gene after recombination between the pair of Cre recombinase recognition sequences. The genetically modified mouse according to claim 1, wherein the Phe residue corresponding to position 74 of the amino acid sequence shown in SEQ ID NO:1 in the TIR1 family protein is replaced with a Gly residue, an Ala residue, or a Ser residue. The genetically modified mouse according to claim 1 or 2, comprising the pair of Cre recombinase recognition sequences, the transcription termination polyA sequence, the TIR1 family gene sequence, and the promoter sequence in the Rosa26 locus. The genetically modified mouse of claim 1, further comprising a gene encoding a target protein to which a degron sequence has been added and/or a gene encoding a Cre recombinase. The genetically modified mouse of claim 4, wherein the target protein is a membrane protein. The genetically modified mouse according to claim 4 or 5, wherein the gene encoding the Cre recombinase is placed under the control of a site-specific promoter. The genetically modified mouse according to claim 6, wherein the function of the gene encoding the target protein at the site where the site-specific promoter drives expression is essential for development and/or survival. 　A sperm, a fertilized egg, or an embryo generated in the genetically modified mouse according to claim 1 or 2. Cells derived from the genetically modified mouse according to claim 1 or 2. A method for inducing degradation of a target protein in a mouse body, comprising:
A degradation induction step of administering an auxin or an auxin analog to a genetically modified mouse to induce degradation of a target protein to which a degron sequence has been added,
The genetically modified mouse comprises a TIR1 family gene expression system, a gene encoding a target protein to which the degron sequence has been added, and a gene encoding a Cre recombinase;
The TIR1 family gene expression system comprises:
A pair of Cre recombinase recognition sequences,
A transcription termination polyA sequence located between the pair of Cre recombinase recognition sequences;
the method comprising: a Transport Inhibitor Response 1 (TIR1) family gene sequence encoding a TIR1 family protein, located downstream of the pair of Cre recombinase recognition sequences; and a promoter sequence located upstream of the pair of Cre recombinase recognition sequences, capable of driving expression of the TIR1 family gene after recombination between the pair of Cre recombinase recognition sequences. The method of claim 10, wherein the dosage of the auxin or auxin analog is between 0.1 mg/kg body weight and 50 mg/kg body weight. The method according to claim 10 or 11, further comprising an evaluation step of evaluating the phenotype of the genetically modified mouse after the degradation induction step. The method according to claim 12, wherein the evaluation step is carried out at least 3 hours after the decomposition induction step. The method according to claim 10 or 11, wherein the genetically modified mouse is a fetus or a preweaned mouse, and the auxin or auxin analog is administered to the mother mouse in the degradation induction step. The method according to claim 10, wherein the genetically modified mouse is a disease model mouse, and the target protein is a candidate protein for drug discovery. The method according to claim 15, wherein the disruption based on a whole-body knockout or conditional knockout of the gene encoding the candidate drug discovery target protein does not show a therapeutic or preventive effect on the disease model mouse, or shows side effects. The method further comprises administering to the genetically modified mouse a drug that targets a factor other than the drug discovery target candidate protein;
The method according to claim 15 or 16, wherein the evaluation step comprises comparing a phenotype obtained when both the candidate drug target protein and the factor are targeted with a phenotype obtained when only the factor is targeted. 1. A method for inducing degradation of a target protein in a cell derived from a mouse, comprising:
A culturing step of culturing the cell according to claim 9 ;
The method described above, further comprising a degradation inducing step of adding an auxin or an auxin analog to the cells to induce degradation of the target protein to which the degron sequence has been added.

Images