JP7812112B2 - Method for producing a non-human animal model of uterine cancer, a non-human animal model of uterine cancer, and a method for evaluating a test substance - Google Patents

Description

The present disclosure relates to a method for producing a non-human animal model of uterine cancer, a non-human animal model of uterine cancer, and a method for evaluating a test substance.

Uterine cancer consists of cervical cancer and uterine cancer. Furthermore, uterine cancer includes endometrial cancer, which originates in the uterine lining, and uterine fibroids, which are cancers that have become malignant in other parts of the body. Endometrial cancer is currently the most common gynecological cancer, and in recent years, the number of young patients has been increasing in particular. For this reason, efforts are underway to develop drugs to treat endometrial cancer.

Uterine cancer model mice are used in the development of therapeutic drugs for uterine cancer. One such uterine cancer model mouse uses a Cre recombinase (Cre)-loxP sequence (Non-Patent Document 1). One such model mouse was created by crossbreeding a mouse that expresses the Cre gene in a uterine tissue-specific promoter-dependent manner with a mouse that contains a loxP-located gene, such as the PTEN (Phosphatase and Tensin Homolog Deleted from Chromosome 10) gene, which is a causative gene for uterine cancer, to knock out the causative gene throughout the uterus. However, uterine cancer develops in epithelial tissue, and mutations in the causative gene in uterine cancers such as endometrial cancer only occur after sexual maturity. The uterine cancer model mouse has the problem of not being able to reproduce the actual disease.

Takiko Daikoku et.al., “Conditional Loss of Uterine Pten Unfailingly and Rapidly Induces Endometrial Cancer in Mice”, 2008, Cancer Res, vol. 68, No. 14, pages 5619-5627

Another uterine cancer model mouse exists, produced by injecting an adenovirus vector (AAV) capable of expressing the Cre gene into the uterus of a mouse carrying a PTEN gene or other gene sandwiched between loxP sequences, thereby knocking out the gene that causes uterine cancer. However, this requires the prior preparation of mice carrying the gene that causes uterine cancer sandwiched between loxP sequences, and is affected by AAV infection, which does not occur in normal uterine cancer, making it difficult to reproduce the actual disease. Furthermore, with all existing models, the genomic DNA of the model animals must be modified in advance, making the creation of the model animals cumbersome.

The present disclosure therefore aims to provide a method for producing a non-human animal model of uterine cancer that can also be produced from wild-type non-human animals.

In order to achieve the above object, the method for producing a non-human uterine cancer model animal of the present disclosure (hereinafter referred to as the "production method") comprises:
a mutation introduction step of contacting genomic DNA of a non-human animal uterine epithelial cell with a nucleic acid editing module to introduce a loss-of-function mutation into a target gene,
The target genes include the PTEN gene and the liver kinase B1 (LKB1) gene.

In the non-human uterine cancer model animal (hereinafter referred to as the "model animal") disclosed herein, the genomic DNA of uterine epithelial cells contains loss-of-function variants of the PTEN gene, the LKB1 gene, and the Tp53 gene.

The method for evaluating a test substance (hereinafter referred to as the "evaluation method") of the present disclosure includes an administration step of administering a test substance to a non-human animal model of uterine cancer;
and evaluating uterine cancer in the non-human uterine cancer model animal,
The non-human animal model of uterine cancer is a non-human animal model of uterine cancer obtained by the method for producing a non-human animal model of uterine cancer according to the present disclosure, and/or the non-human animal model of uterine cancer according to the present disclosure.

According to the present disclosure, non-human animal models of uterine cancer can be produced from wild-type non-human animals.

FIG. 1 is a schematic diagram showing a vector map of gRNA_Cloning Vector BbsI in Example 1. FIG. 2 is a schematic diagram showing a vector map of the pPyCAG-hCas9-IP vector in Example 1. FIG. 3 is a photograph showing the electroporation method in Example 1. FIG. 4 is a schematic diagram of the in vivo genome editing method by electroporation in Example 1. FIG. 5 is a photograph of the uterus observed in Example 1. FIG. 6 shows a photograph of the uterine tissue stained in Example 1. FIG. 7 is a graph showing the relationship between the target gene and the weight of the uterine horn in Example 1. FIG. 8 is a photograph showing the results of the T7 endonuclease I assay in Example 1. FIG. 9 shows photographs of fluorescent immunostaining of uterine epithelial cells in Example 1. FIG. 10 shows photographs of immunohistochemical staining of steroid hormone receptors in the uterus in Example 1.

<Definition>
As used herein, "uterine cancer" refers to cancer that develops in the uterus. Uterine cancer can be further classified according to the location within the uterus where it develops. Specifically, when cancer develops in the endometrium of the uterus, the uterine cancer can also be called "endometrial cancer," and when cancer develops in the cervix of the uterus, the uterine cancer can also be called "cervical cancer."

As used herein, "animal" refers to a human or non-human animal. Furthermore, as used herein, "non-human animal" refers to an animal other than a human. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.

As used herein, "genomic DNA" refers to the deoxyribonucleotides (DNA) in the genome within the nucleus of a eukaryotic cell.

As used herein, "wild-type" means that no exogenous nucleic acid has been introduced into the genomic DNA of the subject animal.

As used herein, "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and/or modified nucleotides. The nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. The nucleic acid may also be referred to as, for example, a "nucleic acid molecule."

As used herein, "nucleic acid editing" means changing the nucleic acid sequence (base sequence) in genomic DNA or creating a state in which a change in the nucleic acid sequence (base sequence) in genomic DNA can occur. The nucleic acid editing may refer to a change in the nucleic acid sequence, or a state in which a change in the nucleic acid sequence can occur due to single-stranding or cutting of the genomic DNA, or a state in which a change in the nucleic acid sequence can occur due to modification of the bases in the genomic DNA.

As used herein, "nucleic acid editing module" refers to a molecule capable of inducing changes in the nucleic acid sequence.

As used herein, "hybridize" refers to annealing with a complementary polynucleotide that occurs due to nucleotide complementarity, specifically, base complementarity in nucleotides, i.e., two polynucleotides can form a non-covalent pair via hydrogen bonds.

As used herein, "complementary" or "complementary" means that nucleotide pairs, i.e., base pairs, can be formed between one polynucleotide and another polynucleotide.

As used herein, "protein" or "peptide" refers to a polymer composed of unmodified (naturally occurring), modified, and/or artificial amino acids.

As used herein, "polypeptide" refers to a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and/or artificial amino acids. A polypeptide is a peptide having a length of 10 amino acids or more.

As used herein, "domain" refers to a structurally or functionally organized region in a protein, polypeptide, and/or peptide.

As used herein, "gene" refers to RNA (e.g., mRNA) that encodes a desired protein or DNA (e.g., cDNA or genomic DNA) that encodes the RNA. The DNA may be double-stranded or single-stranded. As used herein, the gene may also include additional sequences, such as sequences in untranslated regions (UTRs).

As used herein, "regulatory region" refers to a region in genomic DNA that controls gene expression.

As used herein, the term "promoter" or "promoter region" refers to a region that is located upstream of a DNA region encoding a gene or polynucleotide, contains a nucleic acid sequence (base sequence) to which a transcription factor binds, and regulates the amount of transcription of the gene or polynucleotide. The "promoter" or "promoter region" can also be referred to as, for example, a "transcriptional regulatory region."

As used herein, "loss of function" refers to, for example, a state in which the original function of the gene is (significantly) reduced or lost. In other words, as used herein, "loss of function mutation" may refer to either a mutation that (significantly) weakens the original function of the gene, or a mutation that results in complete loss of function. The "mutation that results in complete loss of function" can also be referred to, for example, as a null mutation or amorph.

As used herein, the term "PTEN (Phosphatase and Tensin Homolog Deleted from Chromosome 10) gene" refers to a gene encoding a protein known as a phosphatase (dephosphorylation enzyme) whose main substrate is phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5) _P3 ). PTEN is known to negatively regulate the PI _3- kinase pathway.

As used herein, the "liver kinase B1 (LKB1) gene" is a gene that encodes a protein known as a kinase (phosphorylation enzyme) whose main substrate is AMP kinase. LKB1 is known to be involved in cell polarity and energy metabolism. LKB1 is also known as STK11 (serine/threonine kinase 11).

As used herein, the term "Tp53 (Tumor Protein p53) gene" refers to a tumor suppressor gene that frequently undergoes abnormalities such as point mutations and deletions in cancer.

As used herein, "expression vector" or "vector" means a recombinant plasmid or virus containing a nucleic acid that is delivered to a host cell, either in vitro or in vivo .

As used herein, "exogenous" means introduced into a cell from outside the cells that make up an animal.

As used herein, "isolated" means identified and separated and/or recovered from components in their natural state. The "isolation" can be achieved, for example, by performing at least one purification step.

As used herein, "target site" or "target region" refers to a site or region in genomic DNA intended to induce a desired effect. The desired effect may be, for example, a mutation (e.g., deletion, substitution, insertion, and/or addition) in the nucleic acid sequence.

As used herein, "targeting" means binding to or accumulating in a target region.

As used herein, "positive" means that a higher signal is detected by an analytical method such as immunohistochemistry, which utilizes an antigen-antibody reaction, compared to a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen. As used herein, "negative" means that a signal is detected that is equal to or lower than a negative control reaction using negative control cells that do not express the antigen or an antibody that does not react with the antigen.

The present disclosure will be explained below using examples, but the present disclosure is not limited to the following examples and can be implemented with any modifications. Furthermore, all explanations in this disclosure are mutually applicable unless otherwise specified. Note that, in this specification, the expression "~" is used to include the numerical or physical values before and after it. Furthermore, in this specification, the expression "A and/or B" includes "A only," "B only," and "both A and B."

<Method for producing a non-human animal model of uterine cancer>
In one aspect, the present disclosure provides a method for producing a non-human animal model of uterine cancer that can be produced from a wild-type non-human animal. The method for producing a non-human animal model of uterine cancer of the present disclosure includes a mutation introduction step in which genomic DNA of uterine epithelial cells of the non-human animal is contacted with a nucleic acid editing module to introduce loss-of-function mutations into target genes, the target genes including the PTEN gene and the liver kinase B1 (LKB1) gene. According to the present disclosure, a non-human animal model of uterine cancer can be produced from a wild-type non-human animal. The production method of the present disclosure can improve the likelihood of developing uterine cancer derived from uterine epithelial cells. Therefore, the production method of the present disclosure can also be said to be a method for improving the development of uterine cancer derived from uterine epithelial cells in a non-human animal, for example.

As a result of extensive research, the present inventors came up with the idea that uterine cancer derived from uterine epithelial cells of the non-human animal could be induced by editing the genomic DNA of the uterine epithelial cells using the nucleic acid editing module. The present inventors then discovered that uterine cancer derived from uterine epithelial cells could be induced by introducing a loss-of-function mutation into the target gene in the genomic DNA using the nucleic acid editing module, leading to the establishment of the production method disclosed herein. According to the production method disclosed herein, a loss-of-function mutation is introduced into the target gene in the genomic DNA of the uterine epithelial cells, so prior modification of the genomic DNA of the non-human animal in which uterine cancer is induced is not required. Therefore, according to the production method disclosed herein, a non-human animal model of uterine cancer can be produced from a wild-type non-human animal. Furthermore, the production method disclosed herein is particularly suitable for use in producing a non-human animal model of endometrial cancer from a wild-type non-human animal.

In the mutation introduction step, a nucleic acid editing module is contacted with the genomic DNA of uterine epithelial cells of a non-human animal to introduce a loss-of-function mutation into the target gene. That is, in the mutation introduction step, a loss-of-function mutation is introduced into the target gene in the genomic DNA of the uterine epithelial cells of a non-human animal for some or all of the uterine epithelial cells of the non-human animal. Specifically, in the mutation introduction step, for example, a nucleic acid editing module in the uterine epithelial cells moves into the nucleus of the uterine epithelial cells and performs nucleic acid editing on the target gene in the genomic DNA in the nucleus, thereby introducing a loss-of-function mutation into the target gene. The transport into the nucleus may be, for example, passive transport or active transport. In the latter case, it is preferable that the nucleic acid editing module includes a nuclear localization signal (nuclear transport signal). In the mutation introduction step, the nucleic acid editing module introduces loss-of-function mutations into one or more locations in the target gene. The number of mutations to be introduced in the mutation introduction step can be determined, for example, depending on the location where the loss-of-function mutation is introduced and the degree of loss of function of the target gene. As a result, the mutation introduction step makes it possible to produce non-human animals containing uterine epithelial cells that have a loss-of-function form of the target gene.

The non-human animal may be a wild-type non-human animal, i.e., a non-human animal in which no exogenous nucleic acid has been introduced into its genomic DNA. The wild-type non-human animal can also be referred to as a non-human animal that does not contain the exogenous nucleic acid in its genomic DNA. In the wild-type non-human animal, the genomic DNA of all or some of the cells does not contain the exogenous nucleic acid. When the genomic DNA of some of the cells does not contain the exogenous nucleic acid, it is preferable that the some of the cells are uterine epithelial cells or epithelial cells of the uterine corpus, i.e., endometrial epithelial cells. According to the production method disclosed herein, uterine cancer can be induced even in wild-type non-human animals, as described above.

Examples of the exogenous nucleic acid include a loxP sequence and a sequence encoding Cre recombinase.

The non-human animal may not contain an exogenous loxP sequence in its genomic DNA. The loxP sequence is a 34-bp nucleic acid sequence (SEQ ID NO: 1) derived from bacteriophage P1. The loxP sequence is composed of a Cre binding site nucleic acid sequence with 13-bp nucleic acid sequences symmetrically arranged on both ends and an asymmetric central nucleic acid sequence. The loxP sequence may be a variation of the bacteriophage P1 sequence, and specific examples include the lox511 sequence (SEQ ID NO: 2), the lox2272 sequence (SEQ ID NO: 3), the loxFas sequence (SEQ ID NO: 4), the lox RE sequence (SEQ ID NO: 5), and the lox LE sequence (SEQ ID NO: 6). The loxP sequence is a sequence recognized by Cre recombinase (Cre) derived from bacteriophage P1, resulting in recombination. For the variation sequences, see, for example, References 2 and 3 below.
Reference 2: Robert W Siegel et.al., “Using an in vivo phagemid system to identify non-compatible loxP sequences”, 2001, FEBS Letters, Volume 505, Issue 3, pages 466-466
Reference 3: Kimi Arak et.al., “Targeted integration of DNA using mutant lox sites in embryonic stem cells”, 1997, Nucleic Acids Research, Volume 25, Issue 4, Pages 868-872

loxP sequence (SEQ ID NO: 1)
5'-ATAACTTCGTATAGCATACATTATACGAAGTTAT-3'
lox511 sequence (SEQ ID NO: 2)
5'-ATAACTTCGTATAGtATACATTATACGAAGTTAT-3'
lox2272 sequence (SEQ ID NO: 3)
5'-ATAACTTCGTATAGgATACtTTATACGAAGTTAT-3'
loxFAS sequence (SEQ ID NO: 4)
5'-ATAACTTCGTATAtacctttcTATACGAAGTTAT-3'
lox RE sequence (SEQ ID NO: 5)
5'-ATAACTTCGTATAGCATACATTATACGAAcggta-3'
lox LE sequence (SEQ ID NO: 6)
5'-taccgTTCGTATAGCATACATTATACGAAGTTAT-3'

The target genes are the PTEN gene (Pten) and the LKB1 gene (Lkb1). The production method of the present disclosure preferably further includes the Tp53 gene (Tp53) as one of the target genes. By including the Tp53 gene as one of the target genes, the production method of the present disclosure can produce a non-human animal model of uterine cancer with significantly greater efficiency.

The PTEN gene, the LKB1 gene, and the Tp53 gene are autosomal genes, with each target gene located on both of a pair of autosomes. Therefore, in the mutation introduction step, a loss-of-function mutation may be introduced into the target gene on one of the pair of autosomes, or into the target genes on both of the pair of autosomes; the latter is preferred as it allows for more efficient production of a non-human animal model of uterine cancer. Furthermore, in the mutation introduction step, loss-of-function mutations may be introduced into one, two, or three of the multiple target genes in one of the pair of autosomes, and loss-of-function mutations may be introduced into the remaining target genes in both of the pair of autosomes.

The non-human animal preferably has normal PTEN and LKB1 genes as the target genes, respectively. Furthermore, when the target gene includes the Tp53 gene, the non-human animal preferably has normal PTEN, LKB1, and Tp53 genes as the target genes, respectively. The normal target gene for each target gene can be determined, for example, by referring to nucleic acid sequences registered in a database. Furthermore, in various animals (particularly mammals), the normal target gene for each target gene can be identified using techniques well known in the art based on the nucleic acid sequence of the normal target gene for a given animal. Specifically, the corresponding target gene in a target animal can be found by searching a database containing nucleic acid sequences for the target animal using the nucleic acid sequence of a reference gene for the corresponding target gene (e.g., the PTEN gene, the LKB1 gene, or the Tp53 gene) as a query sequence. Table 1 below shows examples of the accession numbers for the genomic nucleic acid sequences (genes), cDNA nucleic acid sequences (genes (cDNA)), and protein amino acid sequences of the normal mouse PTEN gene (Pten), normal mouse LKB1 gene (Lkb1), and normal mouse Tp53 gene (Tp53).

The normal mouse PTEN gene may be, for example, a polynucleotide consisting of the nucleic acid sequence of SEQ ID NO: 7 below. The protein encoded by the normal PTEN gene may be, for example, a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8 below. The normal mouse PTEN gene may also be a gene encoding a functional equivalent of the PTEN protein of SEQ ID NO: 8.

Nucleic acid sequence of normal PTEN gene (SEQ ID NO: 7, Accession No. 86355519 (NM_008960))
5'- ATGACAGCCATCATCAAAGAGATCGTTAGCAGAAACAAAAGGAGATATCAAGAGGATGGATTCGACTTAGACTTGACCTATATTTATCCAAATATTATTGCTATGGGATTTCCTGCAGAAAGACTTGAAGGTGTATACAGGAACAATATTGATGATGTAGTAAGGTTTTTGGATTCAAAGCATAAAAACCATTACAAGATATACAATCTATGTGCTGAGAGACATTATGACACCGCCAAATTTAAC TGCAGAGTTGCACAGTATCCTTTTGAAGACCATAACCCACCACAGCTAGAACTTATCAAACCCTTCTGTGAAGATCTTGACCAATGGCTAAGTGAAGATGACAATCATGTTGCAGCAATTCACTGTAAAGCTGAAAGGGACGGACTGGTGTAATGATTTGTGCATATTTATTGCATCGGGCAAATTTTTAAAGGCACAAGAGGCCCTAGATTTTTATGGGGAAGTAAGGACCAGAGACAAAAAG -3'

Amino acid sequence of the protein encoded by the normal PTEN gene (SEQ ID NO: 8, Accession No. 6679523 (NP_032986))
MTAIIKEIVSRNKRRYQEDGFDLDLTYIYPNIIAMGFPAERLEGVYRNNIDDVVRFLDSKHKNHYKIYNLCAERHYDTAKFNCRVAQYPFEDHNPPQLEL IKPFCEDLDQWLSEDNHVAAIHCKAGKGRTGVMICAYLLHRGKFLKAQEALDFYGEVRTRDKKGVTIPSQRRYVYYYSYLLKNHLDYRPVALLFHKMMFE TIPMFSGGTCNPQFVVCQLKVKIYSSNSGPTRREDKFMYFEFPQPLPVCGDIKVEFFHKQNKMLKKDKMFHFWVNTFFIPGPEETSEKVENGSLCDQEIDSICSIERADNDKEYLVLTLTKNDLDKANKDKANRYFSPNFKVKLYFTKTVEEPSNPEASSSTSVTPDVSDNEPDHYRYSDTTDSDPENEPFDEDQHSQITKV

An example of a normal mouse LKB1 gene is a polynucleotide consisting of the nucleic acid sequence of SEQ ID NO: 9 below. An example of a protein encoded by the normal LKB1 gene is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10 below. A normal mouse LKB1 gene may also be a gene encoding a functional equivalent of the LKB1 protein of SEQ ID NO: 10.

Nucleic acid sequence of normal LKB1 gene (SEQ ID NO: 9, Accession No. 1690504791 (NM_011492))
5'- ATGGACGTGGCGGACCCCGAGCCGTTGGGCCTTTTCTCCGAGGGCGAGCTGATGTCGGTGGGCATGGACACCTTCATCCACCGCATCGACTCCACCGAGGTAATCTACCAGCCGCGCCGCAAACGCG CCAAGCTCATCGGCAAGTACCTGATGGGGGACCTGCTCGGGGAGGGCTCGTACGGCAAGGTGAAGGAGGTGCTGGACTCCGAGACCTTATGCCGCAGGGCGGTCAAGATCCTCAAGAAGAAAAAAGCTG -3'

Amino acid sequence of the protein encoded by the normal LKB1 gene (SEQ ID NO: 10, Accession No. 7106425 (NP_035622))
MDVADPEPLGLFSEGELMSVGMDTFIHRIDSTEVIYQPRRKRAKLIGKYLMGDLLGEGSYGKVKEVLDSETLCRRAVKILKKKKLRRIPNGEANVKKEIQLLRRLRHRN VIQLVDVLYNEEKQKMYMVMEYCVCGMQEMLDSVPEKRFPVCQAHGYFRQLIDGLEYLHSQGIVHKDIKPGNLLLTTNGTLKISDLGVAEALHPFAVDDTCRTSQGSPA FQPPEIANGLDTFSGFKVDIWSAGVTLYNITTGLYPFEGDNIYKLFENIGRGDFTIPCDCGPPLSDLLRGMLEYEPAKRFSIRQIRQHSWFRKKHPLAEALVPIPPSPD TKDRWRSMTVVPYLEDLHGRAEEEEEEDLFDIEDGIIYTQDFTVPGQVLEEEVGQNGQSHSLPKAVCVNGTEPQLSSKVKPEGRPGTANPARKVCSSNKIRRLSACKQQ

An example of a normal mouse Tp53 gene is a polynucleotide consisting of the nucleic acid sequence of SEQ ID NO: 11 below. Furthermore, an example of a protein encoded by the normal Tp53 gene is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 12 below. The normal mouse Tp53 gene may also be a gene encoding a functional equivalent of the Tp53 protein of SEQ ID NO: 12.

Nucleic acid sequence of normal Tp53 gene (SEQ ID NO: 11, Accession No. 187960038 (NM_011640))
5'-TTTCCCTCCCACGTGCTCCCCTGGCTAAAGTTCTGTAGCTTCAGTTCATTGGGACCATCCTGGCTGTAGGTAGCGACTACAGTTAGGGGGCACCTAGCATTCAGGCCCTCATCCTCCTCCTTCCCAGCAGGGTGTCACGCTTCTCCGAAGACTGG ATGACTGCCATGGAGGAGTCACAGTCGGATATCAGCCTCGAGCTCCCTCTGAGCCAGGAGACATTTTCAGGCTTATGGAAACTACTTCCTCCAGAAGATATCCTGCCATCACCTCACTGCATGGACGATCTGTTGCTGCCCCAGGATGTTGAGGAGTTTTTTGAAGGCCCAAGTGAAGCCCTCCGAGTGTCAGGAGCTCCTGCAGCACAGGACCCTGTCACCGAGACCCCTGGGCCAGTGGCCCCTGCCCCAGCCACTCCATGGCCCCTGTCATCTTTTGTCCCTTCTCAAAAAACTTACCAGGGCAACTATGGCTTCCACCTGGGCTTCCTGCAGTCTGGGACAGCCAAGTCTGTTATGTGCACGTACTCTCCTCCCCTCAATAAGCTATTCTGCCAGCTGGCGAAGACGTGCCCTGTGCAGTTGTGGGTCAGCGCCACACCTCCAGCTGGGAGCCGTGTCCGCGCCATGGCCATCTACAAGAAGTCACAGCACATGACGGAGGTCGTGAGACGCTGCCCCCACCATGAGCGCTGCTCCGATGGTGATG -3'

Amino acid sequence of the protein encoded by the normal Tp53 gene (SEQ ID NO: 12, Accession No. 148747262 (NP_035770))
MTAMEESQSDISLELPLSQETFSGLWKLLPPEDILPSPHCMDDLLLPQDVEEFFEGPSEALRVSGAPAAQDPVTETPGPVAPAPATPWPLSSFVPSQKTYQGNYGFHLGFLQSGTAKSVMCTYSPPLNKLFCQLAKTCPVQLWVSATPPAGSRVRAMAIYKKSQHMTEVVRRCPHHERCSDGDGLAPPQHLIRVE GNLYPEYLEDRQTFRHSVVVPYEPPEAGSEYTTIHYKYMCNSSCMGGMNRRPILTIITLEDSSGNLLGRDSFEVRVCACPGRDRRTEEENFRKKEVLCPELPPGSAKRALPTCTSASPQKKKPLDGEYFTLKIRGRKRFEMFRELNEALELKDAHATEESGDSRAHSSYLKTKKGQSTSRHKKTMVKKVGPDSD

In the mutagenesis step, a mutation is introduced into the target gene so that the original function of the target gene is (significantly) reduced or lost. Therefore, the loss-of-function mutation of the target gene may be, for example, a mutation that results in a (significantly) reduced state of expression of the target gene's mRNA or the protein encoded by the gene, or a mutation that results in a state in which the gene's mRNA or the protein encoded by the gene is not expressed at all, or a mutation that results in a reduced state of expression of functional gene's mRNA or the protein encoded by the gene, or a state in which the functional gene's mRNA or the protein encoded by the gene is not expressed at all.

Examples of the loss-of-function mutation include point mutations, missense mutations, nonsense mutations, frameshift mutations, large deletions of bases over a wide area, and combinations of these. The loss-of-function mutation may, for example, result in the deletion of part or all of each target gene. The frameshift mutation is a mutation that occurs when a base is deleted or inserted, shifting the triplet reading frame (codon). Compared to base pair substitution mutations, the frameshift mutation has a much greater impact on gene function. This is because when the frameshift mutation occurs, the genetic code in the target gene after the point where the frameshift mutation was introduced is significantly shifted, resulting in not only a change in amino acid but also a shift in stop codons, etc.

A loss-of-function form of each target gene can be defined as a gene in which a mutation such as an insertion, deletion, and/or substitution of one or several bases (hereinafter also referred to as "one or more bases") has been introduced into the nucleic acid sequence of a normal target gene of each target gene. As a specific example, the loss-of-function gene (loss-of-function form) of the PTEN gene is a gene in which a mutation such as an insertion, deletion, and/or substitution of one or more bases has been introduced into the nucleic acid sequence of a normal target gene of the PTEN gene. In the loss-of-function form of the PTEN gene, the one or more bases may be, for example, 1 to 1644 bases, 1 to 822 bases, 1 to 411 bases, 1 to 205 bases, 1 to 164 bases, 1 to 102 bases, 1 to 82 bases, 1 to 51 bases, 1 to 41 bases, 1 to 25 bases, 1 to 20 bases, 1 to 12 bases, or 1 to 6 bases. The loss-of-function form of the LKB1 gene is, for example, a gene in which a mutation such as an insertion, deletion, and/or substitution of one or more bases has been introduced into the nucleic acid sequence of a normal target gene of the LKB1 gene. In the loss-of-function form of the LKB1 gene, the one or more bases are, for example, 1 to 514 bases, 1 to 257 bases, 1 to 128 bases, 1 to 64 bases, 1 to 51 bases, 1 to 32 bases, 1 to 25 bases, 1 to 16 bases, 1 to 12 bases, or 1 to 8 bases. The loss-of-function form of the Tp53 gene is, for example, a gene in which a mutation such as an insertion, deletion, and/or substitution of one or more bases has been introduced into the nucleic acid sequence of a normal target gene of the Tp53 gene. In the loss-of-function mutant of the Tp53 gene, the one or more bases are, for example, 1 to 356 bases, 1 to 178 bases, 1 to 89 bases, 1 to 44 bases, 1 to 35 bases, 1 to 22 bases, 1 to 17 bases, 1 to 11 bases, 1 to 8 bases, or 1 to 4 bases. The frameshift mutation occurs, for example, by the insertion or deletion of 3m+1 bases or 3m+2 bases (m is an integer of 0 or greater).

When the target gene is the PTEN gene, the site for introducing the loss-of-function mutation in the PTEN gene can be, for example, the protein coding region, i.e., the exon region or intron region; or a control region such as a promoter region or enhancer region; and is preferably the coding region. As a specific example, when the target gene is the mouse PTEN gene, the site for introducing the loss-of-function mutation is, for example, bases 869 to 1360 in the nucleic acid sequence of SEQ ID NO: 7 (the nucleic acid sequence underlined in the nucleic acid sequence of SEQ ID NO: 7, corresponding to exons 1 to 5).

When the target gene is the LKB1 gene, the site for introducing a loss-of-function mutation in the LKB1 gene may be, for example, the protein coding region, i.e., the exon region or intron region; or a control region such as a promoter region or enhancer region; and is preferably the coding region. As a specific example, when the target gene is the mouse LKB1 gene, the site for introducing the loss-of-function mutation is, for example, bases 824 to 1113 in the nucleic acid sequence of SEQ ID NO: 9 (the nucleic acid sequence underlined in the nucleic acid sequence of SEQ ID NO: 9, corresponding to exon 1).

When the target gene is the Tp53 gene, the site for introducing a loss-of-function mutation in the Tp53 gene can be, for example, the protein coding region, i.e., the exon region or intron region; or a control region such as a promoter region or enhancer region; and is preferably the coding region. As a specific example, when the target gene is the mouse TP53 gene, the site for introducing the loss-of-function mutation is, for example, bases 158 to 707 in the nucleic acid sequence of SEQ ID NO: 11 (the nucleic acid sequence underlined in the nucleic acid sequence of SEQ ID NO: 11, corresponding to exons 1 to 5).

The nucleic acid editing module includes, for example, a nucleic acid sequence recognition unit that recognizes a target site in the target gene and an editing unit that edits the nucleic acid at the target site. If the nucleic acid sequence recognition unit has the function of the editing unit, the nucleic acid editing module may be composed solely of the nucleic acid sequence recognition unit. In the nucleic acid editing module, for example, the nucleic acid sequence recognition module can bind to the target site of the target gene, thereby recruiting (targeting or accumulating) the editing unit to the target site. This allows the nucleic acid editing module to, for example, edit the nucleic acid sequence of the target gene, thereby introducing a loss-of-function mutation into the target gene.

The nucleic acid sequence recognition unit may be, for example, a protein that recognizes a nucleic acid sequence in DNA, or a complex of a protein and a nucleic acid. Specific examples of the nucleic acid sequence recognition unit include proteins that specifically bind to DNA, such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas system, zinc finger motif, transcription activator-like (TAL) effector, PPR (Pentatricopeptide repeat) motif, restriction enzyme, transcription factor, RNA polymerase, and DNA polymerase, or DNA-binding domains thereof. Preferably, the nucleic acid sequence recognition unit is a CRISPR-Cas system, zinc finger motif, TAL effector, or PPR motif, or a DNA-binding domain thereof. For example, when the nucleic acid editing module introduces a loss-of-function mutation by cleaving genomic DNA, the nucleic acid sequence recognition unit preferably cleaves one or both strands of double-stranded DNA, more preferably both strands of double-stranded DNA, to increase the likelihood of altering the nucleic acid sequence of genomic DNA. On the other hand, when the editing unit changes the nucleic acid sequence of genomic DNA to introduce a loss-of-function mutation, for example, the nucleic acid sequence recognition unit preferably does not cleave one or both strands of double-stranded DNA in order to prevent a decrease in the editing efficiency of the editing unit due to cleavage of the genomic DNA, and it is more preferable that the nuclease activity against genomic DNA is inactivated.

Examples of nucleic acid editing modules that have the functions of the editing unit include the CRISPR-Cas system, which includes a Cas protein with nuclease activity, and restriction enzymes.

The CRISPR-Cas system is composed of a Cas protein with nuclease activity and a guide strand (guide RNA) that forms a complex with the Cas protein and hybridizes with the target nucleic acid sequence. When the CRISPR-Cas system comes into contact with the genomic DNA, for example, the Cas9 protein and the guide RNA form a complex.

The Cas protein is not particularly limited, and examples thereof include Cas proteins of types I to V. Specific examples of the Cas protein include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Examples include Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, and Csf4.

The origin of the Cas protein is not particularly limited. When Cas9 is used as the Cas protein, examples of the Cas9 protein include Cas9 (SaCas9) derived from Staphylococcus aureus , Cas9 (SpCas9) derived from Streptococcus pyogenes , and Cas9 (StCas9) derived from Streptococcus thermophilus .

The guide RNA comprises a polynucleotide having a nucleic acid sequence complementary to that of a target gene, and is capable of hybridizing with the target nucleic acid sequence via the polynucleotide. The nucleic acid sequence of the target gene can be appropriately set depending on the site in the target gene where a loss-of-function mutation is to be introduced. Examples of the site in the target gene where a loss-of-function mutation is to be introduced (target site) include the protein coding region in the target gene, i.e., the exon region or intron region; or a regulatory region such as a promoter region or enhancer region. The target site in the nucleic acid sequence of the target gene may be aberrant at one or two sites. When the CRISPR-Cas system is used as the nucleic acid sequence recognition module, the guide RNA is, for example, an RNA molecule comprising a nucleic acid sequence that specifically binds to the nucleic acid sequence of the target gene, i.e., an RNA molecule comprising a polynucleotide complementary to the nucleic acid sequence of the target site in the target gene.

The guide RNA can be appropriately designed depending on the type of Cas protein. The guide RNA may contain only crRNA (CRISPR RNA), or may contain crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA). When the Cas protein is Cas9 protein, the guide RNA may be composed of two RNAs or a single RNA (sgRNA). In the former case, the guide RNA is composed of crRNA and tracrRNA, and the crRNA and tracrRNA hybridize with complementary polynucleotides to form a complex that functions as the guide RNA. In the latter case, the sgRNA is composed of crRNA and tracrRNA, or these are linked via a linker.

The crRNA preferably includes, for example, at its 5' end, a polynucleotide complementary to the nucleic acid sequence of the target gene. By using a nucleic acid sequence in the target region as the target nucleic acid sequence, the crRNA can recruit (target or accumulate) the Cas protein to the nucleic acid sequence in the target region, thereby recruiting (targeting or accumulating) the nucleic acid editing module to the nucleic acid sequence in the target region. The length of the complementary polynucleotide is, for example, 15 to 25 bases long or 18 to 22 bases long.

The length of the guide RNA can be set appropriately depending on, for example, the length of the nucleic acid sequence of the target gene, particularly the length of the nucleic acid sequence of the target site.

The nucleic acid sequence targeted by the guide RNA, i.e., the target site, may be one or two sites. That is, the number of types of guide RNA can be set, for example, depending on the number of target genes and the number of target sites. As a specific example, in a region with a relatively large number of target sites, the guide RNA can encode multiple types of guide RNAs that specifically bind to nucleic acid sequences at different target sites in the target gene, thereby introducing loss-of-function mutations into multiple target genes and/or multiple target sites.

When the nucleic acid editing module includes the nucleic acid sequence recognition unit and the editing unit, the nucleic acid sequence recognition unit can be, for example, a CRISPR-Cas system including a Cas protein with nickase activity or without nuclease activity, the Zinc finger motif, the TAL effector, the PPR motif, a restriction enzyme without nuclease activity, the transcription factor, or a DNA binding domain of the RNA polymerase or DNA polymerase, etc.

When the Cas protein has nickase activity or no nuclease activity, the CRISPR-Cas system includes a Cas protein in which a mutation has been introduced into the domain having nuclease activity of the Cas protein. Specifically, the mutated Cas protein preferably has inactivated nuclease activity (DNA cleavage ability) of at least one of the DNA cleavage domains (cleavage sites) of the Cas protein. As a specific example, the Cas9 protein has an HNH domain and a RuvC domain as DNA cleavage domains. Therefore, it is preferable that at least one of the HNH domain and the RuvC domain of the Cas9 protein is inactivated, and more preferably both are inactivated. The nuclease activity can be inactivated, for example, by introducing an amino acid substitution, a frameshift mutation, and/or a nonsense mutation into the nucleic acid sequence encoding the DNA cleavage domain in DNA encoding the Cas protein. As a specific example, when the Cas9 protein is SpCas9, the HNH domain of the SpCas9 can be inactivated, for example, by substituting the histidine residue (His) at position 840 with an alanine residue (Ala), i.e., the ability to cleave the strand complementary to the guide RNA can be inactivated. Furthermore, the RuvC domain of the SpCas9 can be inactivated, for example, by substituting the aspartic acid residue (Asp) at position 10 with an alanine residue (Ala), i.e., the ability to cleave the strand opposite the strand that forms the complementary strand with the guide RNA can be inactivated. For example, the nuclease activity of the SpCas9 protein can be inactivated by substituting the histidine residue (His) at position 840 with an alanine residue (Ala), and substituting the histidine residue (His) at position 840 with an alanine residue (Ala) (dCas9 protein). The guide RNA can be the same as the guide RNA described above for the Cas protein with nuclease activity.

The zinc finger motif is formed by linking multiple zinc finger units of different C ₂ H ₂ (Cys ₂ His ₂ ) types. One zinc finger unit recognizes a nucleic acid sequence of about 3 bases. The number of zinc finger units can be appropriately determined depending on the nucleic acid sequence of the target region, but is generally 3 to 6. In this case, the zinc finger motif can recognize, for example, a nucleic acid sequence of about 9 to 18 bases. The zinc finger motif can be prepared by known methods such as the modular assembly method (Nat Biotechnol (2002) 20: 135-141), the OPEN method (Mol Cell (2008) 31: 294-301), the CoDA method (Nat Methods (2011) 8: 67-69), or the Escherichia coli one-hybrid method (Nat Biotechnol (2008) 26: 695-701). The zinc finger motif can be prepared by the method described in WO 03/087341, for example.

TAL effectors have a repeating structure of modules consisting of approximately 34 amino acids. The 12th and 13th amino acid residues (repeat variable diresidues: RVD) of each module determine binding stability and nucleic acid (base) specificity. Each module is highly independent, and by arranging modules consecutively, it is possible to create TAL effectors specific to target nucleic acid sequences. TAL effectors specific to target nucleic acid sequences can be designed using open resources such as the REAL method (Curr Protoc Mol Biol (2012) Chapter 12: Unit 12.15), the FLASH method (Nat Biotechnol (2012) 30: 460-465), and the Golden Gate method (Nucleic Acids Res (2011) 39: e82). TAL effectors can be produced, for example, by the method described in WO 2011/072246.

The PPR motif is composed of 35 amino acids and is configured to recognize a specific nucleic acid sequence by arranging a series of PPR motifs that recognize a single nucleic acid (base). The PPR motifs recognize the target nucleic acid (base) at the first, fourth, and second amino acids from the C-terminus (ii(-2)) of each motif. Furthermore, the structure of each motif is independent, and there is no interference from PPR motifs on the C- or N-terminal side. Therefore, by arranging PPR motifs consecutively, a PPR protein specific to a target nucleic acid sequence can be produced. The PPR motif can be produced, for example, by the method described in WO 2014/175284.

When using the DNA-binding domain of the restriction enzyme, transcription factor, RNA polymerase, or DNA polymerase, the DNA-binding domains of these proteins are publicly known, and the DNA-binding domain can be used as the nucleic acid sequence recognition unit.

When the nucleic acid editing module includes the nucleic acid sequence recognition unit and the editing unit, examples of the editing unit include nucleases; base editing enzymes such as cytidine deaminase and adenine deaminase; etc.

The nuclease may be, for example, an endonuclease capable of cleaving one or both strands of the double-stranded genomic DNA. Specific examples include proteins such as FokI endonuclease (Shengdar Q Tsai et al., (2014) Nature Biotech doi:10.1038/nbt.2908) or their nuclease domains.

Examples of the base editing enzyme include AID (Activation-Induced (Cytidine) Deaminase), PmCDA1 (Cytidine deaminase 1), APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like), and ABE (Gaudelli, N. M., et al., Nature, 551, 464-471 (2017)).

In the nucleic acid editing module, the nucleic acid sequence recognition unit and the editing unit are preferably linked directly or indirectly. The direct link means that the nucleic acid sequence recognition unit and the editing unit are directly linked. When the nucleic acid sequence recognition unit and the editing unit are proteins, the direct link means that the protein constituting the editing unit is linked to the N-terminus or C-terminus of the protein constituting the nucleic acid sequence recognition unit. The indirect link means that the nucleic acid sequence recognition unit and the editing unit are linked via a linker. When the nucleic acid sequence recognition unit and the editing unit are proteins, the protein constituting the editing unit is linked via the linker to the N-terminus or C-terminus of the protein constituting the nucleic acid sequence recognition unit. The linker is preferably a peptide linker.

Any sequence can be selected for the linker as long as it does not interfere with the function of the nucleic acid sequence recognition unit and the editing unit. Examples of the linker include a repeat sequence of glycine and serine. The length of the linker is, for example, 5 to 100 amino acids, 5 to 50 amino acids, 10 to 50 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 17 to 30 amino acids, or 22 amino acids. By increasing the length of the linker relatively, the resulting complex can also modify genomic DNA at locations farther from the nucleic acid sequence of the target region. Examples of the linker include GSGSG (SEQ ID NO: 13), GSGGS (SEQ ID NO: 14), SGSGS (SEQ ID NO: 15), or GGGGS (SEQ ID NO: 16), or a sequence consisting of two or three repeats of these; GSGSGGSGSGSGSGGSGGSGSGSGSGSGSG (SEQ ID NO: 17); GSGSGGGSGSGGSGSGGSGGSGGSGGSGSGGSGGSGSGSGGSGGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSG (SEQ ID NO: 18); and the like.

The nucleic acid editing module may be, for example, one type, or two or more types. When there are multiple target genes, the nucleic acid editing module includes, for example, a nucleic acid editing module capable of editing the nucleic acid sequence of each target gene. Therefore, when the target genes in the mutation introduction step are the PTEN gene and the LKB1 gene, the nucleic acid editing module preferably includes a first nucleic acid editing module capable of introducing a loss-of-function mutation into the PTEN gene and a second nucleic acid editing module capable of introducing a loss-of-function mutation into the LKB1 gene. In this case, when a CRISPR-Cas system is used as the nucleic acid editing module, the Cas protein may be one or more types, and the first nucleic acid editing module and the second nucleic acid editing module may be constructed by combining a guide strand specific to the nucleic acid sequence of the PTEN gene with a guide strand specific to the nucleic acid sequence of the LKB1 gene. When the target genes in the mutation introduction step are the PTEN gene, the LKB1 gene, and the Tp53 gene, the nucleic acid editing modules preferably include a first nucleic acid editing module capable of introducing a loss-of-function mutation into the PTEN gene, a second nucleic acid editing module capable of introducing a loss-of-function mutation into the LKB1 gene, and a third nucleic acid editing module capable of introducing a loss-of-function mutation into the Tp53 gene. In this case, when a CRISPR-Cas system is used as the nucleic acid editing module, one or more types of Cas proteins may be used, and the first nucleic acid editing module, the second nucleic acid editing module, and the third nucleic acid editing module may be constructed by combining a guide strand specific to the nucleic acid sequence of the PTEN gene, a guide strand specific to the nucleic acid sequence of the LKB1 gene, and a guide strand specific to the nucleic acid sequence of the Tp53 gene.

The nucleic acid editing module does not contain, for example, Cre recombinase. Because the manufacturing method disclosed herein uses the nucleic acid editing module, it is possible to render the target gene non-functional without using a Cre-loxP sequence, achieving a state similar to that of a target gene knocked out using a Cre-loxP sequence.

The nucleic acid editing module preferably includes a nuclear localization signal (nuclear transport signal). In this case, the nuclear localization signal is preferably linked to, for example, a protein constituting the nucleic acid editing module. As a specific example, when the nucleic acid editing module includes a Cas protein, the nuclear localization signal is preferably linked to at least one of the N-terminus and C-terminus of the Cas protein.

In the mutation introduction step, the nucleic acid editing module may be configured to be able to come into contact with the genomic DNA of the uterine epithelial cells. Therefore, the manufacturing method of the present disclosure may involve introducing the nucleic acid editing module into the uterine epithelial cells and bringing the introduced nucleic acid editing module into contact with the genomic DNA in the uterine epithelial cells. Alternatively, the manufacturing method of the present disclosure may involve introducing a nucleic acid encoding the nucleic acid editing module into the uterine epithelial cells, expressing the nucleic acid editing module from the nucleic acid encoding the nucleic acid editing module, and bringing the expressed nucleic acid editing module into contact with the genomic DNA in the uterine epithelial cells.

The manufacturing method of the present disclosure may include, prior to the mutation introduction step, a step of introducing the nucleic acid editing module from outside the uterine epithelial cells into the uterine epithelial cells (introduction step). If the nucleic acid editing module is composed of multiple components, the introduction step may involve introducing some or all of the components separately from outside the uterine epithelial cells into the uterine epithelial cells, or all of the components may be introduced simultaneously from outside the uterine epithelial cells into the uterine epithelial cells. Furthermore, if multiple components are introduced simultaneously in the introduction step and two or more of the components are capable of forming a complex, the introduction step may involve introducing some or all of the components in the form of a complex from outside the uterine epithelial cells into the uterine epithelial cells. As a specific example, if a CRISPR-Cas system is used as the nucleic acid editing module, the introduction step may involve introducing a Cas protein and a guide strand separately or simultaneously. In the latter case, the introduction step may involve forming a complex between the Cas protein and the guide strand in advance, and then introducing the complex into the uterine epithelial cells from outside the uterine epithelial cells.

In the introduction step, for example, a nucleic acid encoding the nucleic acid editing module may be used as the nucleic acid editing module. In this case, the manufacturing method disclosed herein may further include, for example, a step of expressing the nucleic acid editing module encoded by the nucleic acid (expression step).

When a nucleic acid editing module expressed from a nucleic acid encoding the nucleic acid editing module is used as the nucleic acid editing module, the nucleic acid encoding the nucleic acid editing module is preferably operably linked (inserted) into an expression vector so that the nucleic acid can be expressed. When the nucleic acid editing module is composed of multiple molecules, each molecule constituting the nucleic acid editing module may be inserted into the same expression vector or different expression vectors. As a specific example, when the nucleic acid editing module is composed of a Cas protein and a guide strand, the nucleic acid encoding the Cas protein and the nucleic acid encoding the guide strand may be inserted into the same expression vector or different expression vectors. The expression vector can be prepared, for example, by inserting a nucleic acid encoding the nucleic acid encoding the nucleic acid editing module into a backbone vector (hereinafter also referred to as a "base vector").

The basic vector can be selected appropriately depending on the non-human animal, i.e., the host. The expression vector can be, for example, a non-viral vector such as a plasmid vector, or a viral vector. Examples of plasmid vectors for animals include pCDM8, pMT2PC, pA1-11, pXT1, pRc/CMV, pRc/RSV, and pcDNAI/Neo. Examples of plasmid vectors for plants include vectors containing T-DNA. Examples of viral vectors include retroviruses, vaccinia viruses, and adenoviruses.

The expression vector preferably has a regulatory sequence that regulates the expression of the nucleic acid editing module. Examples of the regulatory sequence include a promoter, terminator, enhancer, polyadenylation signal sequence, and origin of replication (ori). The location of the regulatory sequence in the expression vector is not particularly limited as long as it is located so as to functionally regulate the expression of the nucleic acid editing module, and can be located based on known methods. For example, the regulatory sequence may utilize a sequence already contained in the basic vector, or the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence contained in the basic vector may be replaced with another regulatory sequence.

The expression vector may further include, for example, a coding sequence for a selectable marker. Examples of the selectable marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, and a cell surface receptor marker.

The insertion of nucleic acid (DNA), the insertion of the regulatory sequence, and/or the insertion of the coding sequence of the selection marker into the expression vector may be carried out, for example, using a method using restriction enzymes and ligase, or using a commercially available kit, etc.

In the introduction step, introduction into the uterine epithelial cells can be carried out by, for example, a method for introducing proteins and/or nucleic acids into the cells. Specific examples include electroporation, introduction using a gene gun such as a particle gun, polyethylene glycol, calcium phosphate, lipofection using liposomes, ultrasonic nucleic acid introduction, DEAE-dextran, direct injection using microglass tubes, microinjection, hydrodynamic method, and cationic liposome method.

When the nucleic acid editing module is introduced into the uterine epithelial cells by electroporation, the electroporation method can be described, for example, in Reference 1 below. Specifically, the electroporation method can be performed by, for example, introducing and/or filling the uterus with a solution containing the nucleic acid editing module, and then using a pair of electrodes to apply a voltage to the uterus placed between the electrodes. The voltage application involves, for example, applying a first electric pulse to form pores in the cell membrane of the uterine epithelial cells, and then applying a second electric pulse to introduce the nucleic acid editing module through the pores. The concentration of the nucleic acid editing module in the solution containing the nucleic acid editing module can be set depending on the type of nucleic acid editing module, and is, for example, 0.01 to 100 μmol/L or 0.1 to 10 μmol/L.

As an example, the electroporation method for the non-human animal can be performed as follows. First, in the introduction step, for example, the uterus is exposed from the non-human animal. Next, in the introduction step, for example, the cervix of the uterus is ligated, and then a liquid containing the nucleic acid editing module is filled into the exposed uterus. The filling can be performed using, for example, a syringe. Then, in the introduction step, for example, electrodes are placed at one end and the other end of the uterus so that they are in contact with the uterus, and in this state, a voltage is applied to the electrodes, thereby introducing the nucleic acid editing module from outside the uterine epithelial cells into the uterine epithelial cells.
Reference 1: Ryosuke KOBAYASHI et.al., “A novel method of gene transduction to the murine endometrium using in vivo electroporation”, 2017, J. Vet. Med. Sci., vol. 79, No. 9, pages 1573-1577

The conditions for applying the voltage can be set, for example, depending on the distance between the electrodes and the amount of the nucleic acid editing module introduced. The voltage value for the applied voltage is, for example, -100 to 100 V. The voltage application may be a continuous voltage or a pulsed voltage, with the latter being preferred. When applying a pulsed voltage, pulsed voltages of different voltage values may be applied in the introduction step. In this case, the introduction step preferably includes the steps of applying a first electric pulse (poring pulse) to form pores in the cell membrane of the uterine epithelial cells, applying a second electric pulse (positive transfer pulse) to introduce the nucleic acid editing module through the pores, and applying a third electric pulse (negative transfer pulse) to introduce the nucleic acid editing module through the pores. Each electric pulse may be applied once or multiple times. The voltage value of the first electric pulse is, for example, 10 to 50 V, preferably 10 to 30 V. The voltage value of the second electric pulse is, for example, greater than 0 V and less than or equal to 10 V, preferably 5 to 15 V. The voltage value of the third electric pulse is, for example, less than 0 V and greater than or equal to -30 V, preferably -15 to -5 V. The application time of each pulse voltage (electric pulse) is, for example, 1 to 200 ms.

When the nucleic acid editing module is introduced into the uterine epithelial cells using the polyethylene glycol method, the calcium phosphate method, or the lipofection method, the introduction step can be carried out, for example, by introducing or filling the uterus with a liquid containing the nucleic acid editing module and maintaining this state for a predetermined period of time. The predetermined period of time is, for example, 1 to 72 hours or 6 to 48 hours. Furthermore, in the introduction step, the liquid containing the nucleic acid editing module may be removed from the uterus after the maintenance, for example.

The production method of the present disclosure may include, after the introduction step, a step of selecting a non-human animal into which a loss-of-function mutation has been introduced into the target gene (selection step). In this case, the selection step can be performed, for example, by subjecting the non-human animal to the following steps (a) and (b):
(a) a detection step of detecting whether the target gene in the non-human animal has lost its function; (b) a mutant selection step of selecting the non-human animal as a candidate non-human model animal for uterine cancer if the target gene has lost its function.

When the selection step includes steps (a) and (b), the selection step may be carried out using, for example, the nucleic acid sequence of each target gene as an index, or the expression level of each target gene as an index.

When the nucleic acid sequence of each target gene is used as an indicator, in step (a), the loss of function of each target gene may be detected by, for example, decoding the nucleic acid sequence of each target gene of the non-human animal and comparing it with the nucleic acid sequence of the corresponding normal target gene. The nucleic acid sequence can be decoded, for example, using a sequencer. Then, in step (b), for example, when the nucleic acid sequence of each target gene of the non-human animal is a nucleic acid sequence in which a loss-of-function mutation has been introduced relative to the nucleic acid sequence of the corresponding normal target gene, the non-human animal is selected as the candidate non-human model animal for uterine cancer. The selection conditions are described below. The nucleic acid sequence of each target gene described above can be referenced for the nucleic acid sequence of the normal target gene. The comparison of the nucleic acid sequences can be performed, for example, using nucleic acid sequence analysis software (e.g., the aforementioned BLAST). In step (b), the region for nucleic acid sequence comparison may be the intron region of each target gene or the exon region of each target gene, with the latter being preferred. Furthermore, if the loss of function of each target gene is caused by introducing a mutation, such as an insertion, deletion, and/or substitution of one or more bases, into the nucleic acid sequence of the corresponding normal target gene, step (a) may be carried out, for example, using a primer set, probe, or a combination thereof that can detect at least one mutation. The primer set and probe can be designed, for example, using known design methods based on the type of mutation.

In step (b), for example, if one or more bases have been inserted, deleted, and/or substituted relative to the normal target gene of each target gene, the target gene may be determined to be a loss-of-function gene. Also, in step (b), for example, if a frameshift mutation has been introduced into the normal target gene of each target gene, the target gene may be determined to be a loss-of-function gene. In step (b), for example, if the normal target gene of each target gene is partially or completely deleted, the target gene may be determined to be a loss-of-function gene.

In step (b), selection may further be based on the genotype of each target gene. Specifically, in step (b), if the genotype of each target gene corresponds to a heterozygote (zygotic type) or homozygote (homozygous type) for a loss-of-function form of each target gene, the non-human animal may be selected as the candidate non-human model animal for uterine cancer. Specifically, in step (b), for example, if the non-human animal has a heterozygote or homozygote for a loss-of-function form of the PTEN gene and the LKB1 gene, respectively, the non-human animal may be selected as the candidate non-human model animal for uterine cancer. Furthermore, in step (b), for example, if the non-human animal has a heterozygote or homozygote for a loss-of-function gene for the PTEN gene, the LKB1 gene, and the Tp53 gene, the non-human animal may be selected as the candidate non-human model animal for uterine cancer.

When the expression level of each target gene is used as an indicator, in step (a), the loss of function of each target gene may be detected, for example, by detecting the mRNA of each target gene or the function of the protein encoded by each target gene in the non-human animal. Furthermore, in step (a), the loss of function of each target gene may be detected, for example, by detecting the presence or absence of expression of each target gene or the protein encoded by each target gene in the non-human animal, or by detecting the expression level of each target gene or the protein encoded by each target gene.

When the selection step is based on the expression of each target gene or the protein encoded by each target gene, in step (a), for example, the expression level of at least one of each target gene and the protein encoded by each target gene in a biological sample from the non-human animal is measured. Then, in step (b), non-human animals having a loss-of-function form of each target gene are selected based on the expression level of at least one of each target gene and the protein encoded by each target gene in the biological sample from the non-human animal and a reference value. Specifically, in step (b), selection of non-human animals having a loss-of-function form of each target gene can be carried out by, for example, comparing the expression level of at least one of each target gene and the protein encoded by each target gene in the biological sample from the non-human animal with the reference value.

The biological sample from the non-human animal is not particularly limited and may be, for example, the uterus of the non-human animal or a sample derived from the uterus. The type of biological sample used in step (a) may be, for example, one type, or two or more types.

In step (a), the expression level of each target gene can be measured by, for example, semi-quantitative PCR, quantitative PCR, Northern blotting, digital PCR, RNA sequence analysis (RNAseq), etc. In addition, in step (a), the expression level of the protein encoded by each target gene can be measured by, for example, a spectrophotometer-based method such as ultraviolet absorption or the bicinchoninic acid method, or a protein quantification method such as ELISA or Western blotting.

Examples of the reference value include the expression level of each target gene or the protein encoded by each target gene in a non-human animal having normal target genes, and the expression level of each target gene or the protein encoded by each target gene in a non-human animal having a loss-of-function form of a normal target gene corresponding to each target gene of interest (e.g., a non-human animal completely deficient in the PTEN gene, LKB1 gene, and/or Tp53 gene). When the expression level of each target gene in a non-human animal having a loss-of-function form of each target gene is used as the reference value, the non-human animal having a loss-of-function form of each target gene may be, for example, a non-human animal having a loss-of-function form of either one of the two PTEN genes, LKB1 genes, and/or Tp53 genes located on each of a pair of chromosomes, or a non-human animal having a loss-of-function form of both target genes. The expression level of each target gene or the protein encoded by each target gene used as the reference value can be obtained, for example, by measuring the expression level of each target gene or the protein encoded by each target gene in a biological sample collected under the same conditions as the biological sample from the non-human animal, using the same method as for the biological sample from the non-human animal. The reference value may be measured, for example, in advance, or may be measured simultaneously with the biological sample from the non-human animal.

In this case, in step (b), the method for assessing whether each target gene in the non-human animal is loss-of-function is not particularly limited and can be determined appropriately depending on the type of reference value. Specifically, if the expression level of each target gene in the biological sample from the non-human animal is lower than the expression level of the corresponding gene in a non-human animal having a normal target gene, is the same as the expression level of the target gene in a non-human animal having a loss-of-function form of the target gene (if there is no significant difference), and/or is lower than the expression level of the gene in a non-human animal having a loss-of-function form of the target gene, the non-human animal can be assessed as having, for example, the target gene. Furthermore, if the expression level of a protein encoded by each target gene in the biological sample from the non-human animal is lower than the expression level of the protein encoded by the corresponding gene in a non-human animal having a normal target gene, the non-human animal can be evaluated as having a loss-of-function form of the target gene, for example, if the expression level of the protein encoded by the target gene is the same (if there is no significant difference) as in a non-human animal having a loss-of-function form of the target gene, and/or if the expression level of the protein encoded by the gene in a non-human animal having a loss-of-function form of the target gene is lower than the expression level of the protein encoded by the gene in a non-human animal having a loss-of-function form of the target gene. In step (b), a non-human animal evaluated as having a loss-of-function form of the PTEN gene and the LKB1 gene, or the PTEN gene, the LKB1 gene, and the Tp53 gene, for example, is selected as the candidate non-human model animal for uterine cancer.

In step (b), the genotype of each target gene may be evaluated, for example, based on the expression level of each target gene. Specifically, the evaluation may involve determining whether the animal is homozygous for a normal target gene, heterozygous for a normal target gene and a loss-of-function gene, or homozygous for a loss-of-function gene. In this case, the reference values may be determined using a non-human animal having a normal target gene (normal non-human animal), a non-human animal in which one of two genes located on each of a pair of chromosomes is a normal target gene and the other is a loss-of-function gene (heterozygous non-human animal), and/or a non-human animal in which both of two genes located on each of a pair of chromosomes are loss-of-function genes (homozygous non-human animal for a loss-of-function gene). Specifically, in step (b), if the expression level of a target gene in a non-human animal is equivalent to the expression level of the target gene in a normal non-human animal, a heterozygous non-human animal, or a homozygous loss-of-function gene, the non-human animal can be evaluated as having the same genotype as a non-human animal with an equivalent expression level.

Furthermore, in step (b), selection may be based on evaluation of the obtained genotype of each target gene. As a specific example, in step (b), if the genotypes of the target genes correspond to the above-mentioned genotype combinations, the non-human animal may be selected as the candidate non-human model animal for uterine cancer. As a specific example, in step (b), the non-human animal may be selected as the candidate non-human model animal for uterine cancer if, for example, it has loss-of-function forms in the PTEN gene and the LKB1 gene in a heterozygote or homozygote form. Furthermore, in step (b), the non-human animal may be selected as the candidate non-human model animal for uterine cancer if, for example, it has loss-of-function forms in the PTEN gene, the LKB1 gene, and the Tp53 gene in a heterozygote or homozygote form.

The manufacturing method of the present disclosure may include, after the mutation introduction step, a step of growing the obtained non-human animal and causing uterine cancer to form (formation step). The period of the formation step may be any period during which uterine cancer can be formed, such as 14 days to 4 months, or 2 to 3 months. The rearing conditions for the non-human animal in the formation step can be set according to the conditions of the non-human animal. The uterine cancer of a non-human animal obtained by the manufacturing method of the present disclosure may be female hormone receptor-positive or -negative. Examples of the female hormone receptor include estrogen receptor and progesterone receptor. The uterine cancer may be progesterone receptor-negative and/or estrogen receptor-positive, for example.

Sequence information for the proteins, fusion proteins, or nucleic acids (e.g., DNA or RNA) encoding them described herein is available from Protein Data Bank, UniProt, GenBank, or the like. Furthermore, RNA nucleic acid sequences can also be obtained from the corresponding DNA nucleic acid sequences using appropriate sequence conversion software. DNA encoding the nucleic acid editing module described herein may be obtained by cloning from mRNA using molecular biology methods, or by chemically synthesizing DNA based on the sequence information. Furthermore, when obtaining the DNA, codon optimization may be performed to suit the non-human animal (host) into which the nucleic acid will be introduced. This can be expected to increase the amount of protein expressed in the host, for example. Data on codon usage in the host to be used may be obtained, for example, from the genetic code usage database published on the website of the Kazusa DNA Research Institute (http://www.kazusa.or.jp/codon/index.html), or references listing codon usage in each host may be referenced.

<Non-human animal model of uterine cancer>
In another aspect, the present disclosure provides a non-human uterine cancer model animal that has the potential to develop uterine cancer. In the non-human uterine cancer model animal of the present disclosure, the genomic DNA of uterine epithelial cells comprises a loss-of-function mutant of the PTEN gene, a loss-of-function mutant of the LKB1 gene, and a loss-of-function mutant of the Tp53 gene. The model animal of the present disclosure has the potential to develop uterine cancer derived from uterine epithelial cells. The model animal of the present disclosure can be suitably used, for example, for screening candidate therapeutic agents for uterine cancer.

The model animal may have a loss-of-function mutant of the PTEN gene, a loss-of-function mutant of the LKB1 gene, and a loss-of-function mutant of the Tp53 gene, and the genotype of each gene may be heterozygous, in which one gene on a pair of chromosomes is normal and the other is loss-of-function, or may be homozygous, in which both genes located on each of a pair of chromosomes are loss-of-function, preferably the latter.

In the model animal, for example, it is preferable that the genomic DNA of the uterine epithelial cells does not contain loxP sequences.

In the model animal, the uterine epithelial cells are preferably epithelial cells of the uterine corpus, i.e., endometrial epithelial cells. In this case, the non-human uterine cancer model animal of the present disclosure can also be referred to as an endometrial cancer model non-human animal.

The tissues from which uterine cancer originates in the model animals can be evaluated using, for example, histological staining, immunohistological staining using epithelial cell markers, etc.

The non-human animals disclosed herein may not contain a loss-of-function form of the Tp53 gene.

<Method for evaluating test substances>
In another aspect, the present disclosure provides a method for evaluating whether a test substance can be a candidate substance for preventing or treating uterine cancer. The test substance evaluation method of the present disclosure includes an administration step of administering the test substance to a non-human animal model of uterine cancer, and an evaluation step of evaluating uterine cancer in the non-human animal, wherein the non-human animal model of uterine cancer is a non-human animal model of uterine cancer obtained by the production method of the non-human animal model of uterine cancer of the present disclosure and/or the non-human animal model of uterine cancer of the present disclosure. The evaluation method of the present disclosure allows evaluation of whether the test substance can be a candidate substance for preventing or treating uterine cancer.

In the administration step, the test substance may be administered, for example, before or after the induction of uterine cancer.

In the evaluation step, uterine cancer can be evaluated, for example, by evaluating the pathology of the model animal. Specific examples include evaluating the presence or absence or severity of uterine cancer onset, the presence or absence or severity of enlargement or shrinkage of uterine cancer, and the growth of uterine cancer. The evaluation step also includes a selection step in which the test substance that inhibits the onset of uterine cancer, shrinks uterine cancer, and/or inhibits the growth of uterine cancer is selected as a candidate substance for preventing or treating uterine cancer.

The type of test substance may be any substance that can be a candidate for a preventive or therapeutic drug. Examples of test substances include proteins, antibodies, peptides, nucleic acid molecules, sugar chains, lipids, low molecular weight organic compounds, low molecular weight inorganic compounds, bacterially released substances, fermentation products, cell extracts, cell culture supernatants, plant extracts, and animal tissue extracts. The test substance may be of one type or multiple types.

The evaluation method of the present disclosure may include, prior to the administration step, a production step of producing the non-human animal uterine cancer model using the production method of the present disclosure.

The present disclosure will be explained in detail below using examples, but the present disclosure is not limited to the aspects described in the examples. Unless otherwise specified, commercially available reagents, kits, etc. were used according to their protocols.

[Example 1]
It was confirmed that uterine cancer can be induced by the manufacturing method disclosed herein.

Using genome editing technology (CRISPR-Cas9 system) and in vivo electroporation technology, we confirmed that endometrial cancer can be induced from wild-type mice by introducing loss-of-function mutations into target genes.

(1) Genome editing technology The crRNA or gRNA sequence was designed using CRISPRDirect (https://crispr.dbcls.jp/) and CRISPOR (http://crispor.tefor.net/). Specifically, Pten, Tp53, and Lkb1 were used as target genes. The gRNA target sequences selected from each of the target genes were cloned into gRNA_Cloning Vector BbsI (Addgene, Cat. No. 128433) shown in Figure 1. The target sequences of crRNA or gRNA are shown in Table 2. The crRNA, tracrRNA, and High fidelity SpCas9 (Sp HiFi Cas9 Nuclease V3) were purchased from Integrated DNA Technologies.

For each target sequence, equal amounts of crRNA and tracrRNA were mixed in duplex buffer (Integrated DNA Technologies) and heated at 95°C for 5 minutes. After heating, the mixture was left to stand at room temperature (approximately 25°C; the same applies below) for 10 minutes to obtain a complex (hereinafter referred to as "crRNA/tracrRNA").

The Cas9 expression plasmid pPyCAG-hCas9-IP shown in Figure 2 was constructed by inserting human codon-optimized Cas9 (hCas9) into the BstXI site of the pPyCAG-BstXI-IP vector (provided by RIKEN).

(2) Preparation of Uterine Cancer Model Mice Using the RNP Method For the uterine cancer model mice using the RNP method, virgin female mice (strain: ICR, sold by Japan SLC) aged 6 weeks or older were used. Three types of anesthetics were intraperitoneally administered to the mice. The anesthetics used were medetomidine hydrochloride, midazolam, and butorphanol tartrate. After anesthesia, the uterine horns were externalized, and the cervix was sutured to prevent leakage of the injected reagent. Next, equal amounts of a solution containing Cas9 protein (500 ng/μL) and a solution containing crRNA/tracrRNA (3 μmol/L each) were mixed, and 5 μL of the resulting mixture (genome editing solution) (Cas9: 250 ng/μL, crRNA/tracrRNA: 1.5 μmol/L each) was injected into the endometrial cavity from the fallopian tube side. In addition, the genome editing solution forms a complex (RNP) between the Cas9 protein and crRNA/tracrRNA. Immediately after the injection, as shown in Figure 3, the uterine horn was clamped between platinum plate electrodes (CUY650P5, manufactured by NEPA GENE). In the control group (Control), the same procedure was performed except that an equal volume of OptiMEM medium was used instead of the genome editing solution.

Figure 4 shows a schematic diagram of the in vivo genome editing method using electroporation. As shown in Figure 4(A), the uterine horn was clamped between platinum plate electrodes, and three sets of electric pulses were applied using a NEPA21 electroporator (NEPA GENE). After the electric pulses, the electrode direction was changed and three more sets of electric pulses were applied, as shown in Figure 4(B). This resulted in RNP delivery into the uterine epithelial layer and loss-of-function mutations in Pten, Lkb1, and Tp53. Each set of electric pulses consisted of a poring pulse (PP) and a transfer pulse (TP). The PP conditions were: voltage 20 V, pulse length 30 ms, pulse interval 50 ms, number of pulses 3, decay rate 10%, and positive polarity. The TP conditions were: voltage 10 V, pulse length 50 ms, pulse interval 50 ms, number of pulses 3, decay rate 40%, and positive/negative polarity. Specifically, the electroporation consisted of one set of poring pulses (PP), one set of positive voltage transfer pulses (TP), and one set of negative voltage transfer pulses (TP). As a control, electroporation was performed on a different site in the uterus of the same mouse, except that physiological saline was used instead of the genome editing solution. The inventors of the present application confirmed that when a vector capable of expressing GFP was introduced into the uterus by electroporation, GFP expression was observed only in the epithelial layer, but not in the interstitial or muscle tissue. This is presumably because the expression vector cannot cross the basement membrane of the epithelial layer and migrate to the interstitial and muscle tissues using electroporation.

(3) Preparation of uterine cancer model mice using the plasmid method Preparation of uterine cancer model mice using the plasmid method was carried out in the same manner as in Example 1(2) above, except that the genome editing solution containing Cas9 and crRNA/tracrRNA was replaced with the genome editing solution under the following conditions (1) or (2).
Condition (1):
pPyCAG-hCas9-IP: 352 μmol/l
gRNA vector for each target gene: 117 μmol/L
Condition (2):
pPyCAG-hCas9-IP: 76 μmol/l
gRNA vector for each target gene: 208 μmol/L

(4) Observation of the Presence or Absence of Uterine Tumors The mice obtained in Examples 1(2) and (3) above were examined for the presence or absence of uterine tumors 4 months after the electric pulse treatment. Specifically, the treated mice were dissected and observed for the presence or absence of tumors. The results for the mice obtained in Example 1(2) above are shown in Figure 5.

Figure 5 is a photograph of the uterus. As shown in Figure 5, it was found that tumors formed at the site where the genome editing solution was injected and electroporation was performed after the injection. Furthermore, no tumors formed at the site where physiological saline was injected and electroporation was performed after the injection. The same was true for the mice obtained in Example 1 (3). These results demonstrate that by combining genome editing and in vivo electroporation to introduce loss-of-function mutations into the three genes Pten, Lkb1, and Tp53, it is possible to introduce external nucleic acids locally to the site where electroporation was performed and induce tumor formation.

(5) Investigation of the tissue origin of uterine tumors. It was confirmed whether the tumors formed in the uterus in Example 1(4) were derived from epithelial tissue. Specifically, tumors were collected from the uterine body and fixed using 4% paraformaldehyde. After fixation, the uterine body was embedded in paraffin to prepare 3 μm-thick paraffin-embedded sections. HE staining was performed on the obtained paraffin-embedded sections using a standard method. After staining, pathological diagnosis of the uterine body was performed on each section using an optical microscope (BZ-9000, Keyence Corporation). These results are shown in Figure 6.

Figure 6 shows a photograph of uterine tissue stained with histological staining. As shown in Figure 6, no tumors were formed in the control group (Control). On the other hand, tumors were observed in mice injected with the genome editing solution and then electroporated (Tumor (Pten + Tp53 + Lkb1)). Furthermore, pathological diagnosis revealed that the tumors were derived from epithelial tissue.

These results demonstrate that the in vivo in utero electroporation method disclosed herein can deliver exogenous nucleic acids exclusively to mouse endometrial epithelial cells, and can induce tumors by introducing loss-of-function mutations in the three genes Pten, Lkb1, and Tp53 in uterine epithelial cells.

(6) Study of Tumor Formation Efficiency The RNP method and the plasmid method are known as methods for introducing nucleic acid editing modules for genome editing. The RNP method is a method in which a complex between a Cas protein and a guide RNA is formed and introduced into cells to perform genome editing. On the other hand, the plasmid method is a method in which a plasmid encoding a Cas protein and a plasmid encoding a guide RNA are introduced into cells to perform genome editing. Therefore, we investigated which method has a higher efficiency of introducing loss-of-function mutations. Specifically, we examined the rate of endometrial tumor development three months after treatment for mice treated with the RNP method of Example 1 (2) and the plasmid method of Example 1 (3). The conditions and results are shown in Table 3 below.

Table 3 shows the introduction method, conditions, number of samples, number of tumors formed, and rate of tumor formation. As shown in Table 3, under condition (1) using the plasmid method, the rate of endometrial cancer formation was 10%, and under condition (2) using the plasmid method, the rate of endometrial cancer formation was 50%. In contrast, under condition (3) using the RNP method, the rate of endometrial cancer formation was 70%, which was higher than under conditions (1) and (2) using the plasmid method. These results demonstrate that, in the manufacturing method disclosed herein, the RNP method, as a method for introducing a nucleic acid editing module, can more efficiently introduce loss-of-function mutations into genomic DNA than the plasmid method.

(7) Target Gene Study The combination of target genes required for the induction of endometrial cancer was studied. Specifically, a mixture of crRNA/tracrRNA was prepared for different combinations of target genes. Then, mice were treated in the same manner as in the RNP method of Example 1 (2), except that the genome editing solution was used to prepare the mixture to search for the combination of target genes required for the induction of endometrial cancer. Then, for each mouse, the incidence of tumors was examined by evaluating whether macroscopic lesions, endometrial cancer, or squamous (stratified) epithelialization occurred in the endometrium three months after the treatment.

After 3 months, uterine horns were harvested from the mice and weighed. The uterine horns were then fixed in 4% paraformaldehyde-containing phosphate buffered saline (PBS). After fixation, the uterine horns were cut into 2-4 pieces and embedded in paraffin blocks to prepare 3 μm-thick paraffin-embedded sections. The resulting sections were stained with HE and subjected to histological analysis. For histological analysis, 12 sections were analyzed and evaluated for each sample. Each section was separated by a distance of at least 100 μm. The macroscopic lesions were evaluated based on the number of tumors confirmed at the time of dissection.

Endometrial cancer was assessed by confirming significant destruction of normal endometrial structure and invasion into the myometrium. Endometrial cancer was assessed by counting the number of tumors identified on tissue sections with myometrial invasion. To prepare each section for immunostaining analysis, the sections were deparaffinized and autoclaved (121°C, 5 minutes) in the presence of 10 mmol/L sodium citrate buffer (pH 6.0) to activate the antigen. After blocking with 3% bovine serum albumin in PBS supplemented with Tween® 20, primary antibody dilutions were applied and incubated overnight (approximately 8-10 hours) at 4°C. The primary antibodies used were PGR (1:400 dilution, ab101688, Abcam) and ESR (1:100 dilution, ab75635, Abcam). Next, the sections after incubation were reacted with Histofine Simple Stain MAX-PO (registered trademark) (manufactured by Nichirei) at room temperature for 30 minutes. Signals were developed using 3-3'-diaminobenzidine. HE staining was used as a counterstain. Squamous epithelialization was evaluated based on whether the epithelium was stratified. Squamous epithelialization was evaluated by counting the number of areas where squamous epithelialization was confirmed on the tissue sections. The conditions and results are shown in Table 4 and Figure 7 below.

Table 4 shows the target gene combinations, number of samples, number of tumors formed by type, and rate of tumor formation. As shown in Table 4, when the target gene combination consisted of one gene, the tumor formation rate was 20% only in the case of Lkb1, and 0% for other genes. Furthermore, when the target gene combination consisted of two genes, only in the case of the combination of Pten and Lkb1, the tumor formation rates were 44% for macroscopic lesions, 56% for endometrial cancer, and 22% for squamous cell transformation, and 0% for other combinations. On the other hand, when the target gene combination consisted of three genes, the tumor formation rates were 70% for macroscopic lesions, 90% for endometrial cancer, and 50% for squamous cell transformation.

Figure 7 is a graph showing the relationship between target genes and uterine horn weight. In Figure 7, the horizontal axis indicates the type of target gene, and the vertical axis indicates uterine horn weight. As shown in Figure 7, when Pten and Lkb1 were the target genes, some individuals showed an increase in uterine horn weight, and when Pten, Lkb1, and Tp53 were the target genes, the number of individuals showing an increase in uterine horn weight further increased. Furthermore, as shown in Figure 7, an increase in uterine horn weight increased the number of individuals with stratified endometrium.

These results show that when the target gene combination is Pten and Lkb1, endometrial cancer can be induced efficiently, and that by targeting Tp53, endometrial cancer can be induced even more efficiently. Furthermore, it was found that by targeting three genes, it is possible to induce uterine cancer with a more malignant stage.

(8) Confirmation of Introduction of Loss-of-Function Mutations into Target Genes Individuals with uterine cancer were examined for the introduction of loss-of-function mutations into tumor target genes. Specifically, a T7 endonuclease I assay was used to examine the introduction of mutations into tumor target genes. Endometrial cancer model mice prepared in Example 1 (2) or (3) were dissected 3 months after treatment to obtain endometrial cancer. The genome editing target site was amplified from the endometrial cancer genomic DNA by PCR. The resulting PCR product (100 ng) was denatured at 95°C for 5 minutes. After denaturation, the PCR product denatured in NEBuffer 2 (NEB) was gradually cooled and reannealed. The reannealed PCR product was digested with 5 units of T7 endonuclease I (NEB) at 37°C for 15 minutes. After the digestion, 0.75 μl of 250 mmol/L EDTA was added to the reaction solution to terminate the digestion reaction. The digestion pattern of the PCR product was evaluated using capillary and microchip electrophoresis (MCE-202 MultiNA, Shimadzu Corporation) (Tu). A control was evaluated in the same manner except that physiological saline was used (Wt). These results are shown in Figure 8.

Figure 8 is a photograph showing the results of the T7 endonuclease I assay. As shown in Figure 8, in the control (wild-type tissue), no digestion bands were observed for all target genes, Tp53, Pten, and Lkb1. On the other hand, in endometrial cancer (Tumor), digestion bands were detected for all target genes, Tp53, Pten, and Lkb1.

These results demonstrate that introducing a nucleic acid editing module into uterine epithelial cells using in vivo in utero electroporation introduces loss-of-function mutations into the genomic DNA of the uterine epithelial cells, resulting in the development of uterine cancer.

(9) Histological Study of Triple Mutant Endometrial Cancer To determine whether the uterine tumors formed in Example 1(7) were derived from epithelial tissue, fluorescent immunostaining was performed. The tumors were derived from individuals with loss-of-function mutations in all target genes, Tp53, Pten, and Lkb1. Specifically, tumors from the uterine corpus were harvested and fixed using 4% paraformaldehyde. After fixation, the uterine corpus was embedded in paraffin to prepare 3-μm-thick paraffin-embedded sections. The sections were deparaffinized and then autoclaved in 10 mmol/L sodium citrate buffer (pH 6.0) at 121°C for 5 minutes to activate the antigen. Subsequently, blocking was performed using 3% BSA-containing PBS supplemented with Tween® 20. After blocking, the sections were incubated with a primary antibody overnight at 4°C, followed by staining with a secondary antibody at 23°C for 1 hour. The primary antibodies used for staining epithelial cells were rat anti-CK8 antibody (50x dilution, Troma-I, provided by the University of Iowa), and mouse anti-αSMA antibody (1000x dilution, Cat. No. ab7817, Abcam) for staining smooth muscle cells. The secondary antibodies used were Alexa Fluor 488-labeled goat anti-rat IgG(H + L) (500x dilution, Cat. No. ab150157, Abcam) and Alexa Fluor 594-labeled goat anti-mouse IgG(H + L) (500x dilution, Cat. No. ab150116, Abcam). Nuclei were stained with VECTASHIELD Hard Set Mounting Medium with DAPI (Vector Laboratories). After staining, each section was subjected to pathological diagnosis of the uterine corpus using an optical microscope (BZ-9000, manufactured by Keyence Corporation) and ImageJ software. The results are shown in FIG.

Figure 9 is a photograph of fluorescent immunostaining of uterine epithelial cells. In Figure 9, the scale bar indicates 100 μm. In Figure 9, the white dashed line indicates the border between the endometrial stroma and the smooth muscle layer. As shown in Figure 9, in the control, epithelial markers were observed on the endometrial stroma side but not on the smooth muscle layer. On the other hand, in endometrial cancer, epithelial markers were observed not only on the endometrial stroma side but also on the smooth muscle layer side. From the above, it was found that epithelial cells had infiltrated into the smooth muscle layer in endometrial cancer in the uterine cancer model mice obtained by the method of the present disclosure.

These results demonstrate that triple mutant endometrial cancer, in which loss-of-function mutations have been introduced into all target genes of Tp53, Pten, and Lkb1, as disclosed herein, is a tumor derived from epithelial tissue.

(10) Investigation of Hormone Receptors in Triple Mutant Endometrial Cancer To confirm the expression of sex steroid hormone receptors in the uterine tumors formed in Example 1(7), immunohistochemical staining for the hormone receptors progesterone receptor (PR) and estrogen receptor (ER) was performed. The tumors were derived from individuals with loss-of-function mutations in all target genes, Tp53, Pten, and Lkb1. Specifically, tumors from the uterine body were harvested and fixed using 4% paraformaldehyde. After fixation, the uterine body was embedded in paraffin and 3 μm-thick paraffin-embedded sections were prepared. After deparaffinization, the sections were autoclaved in 10 mmol/L sodium citrate buffer (pH 6.0) at 121°C for 5 minutes to activate the antigen. Blocking was then performed using PBS containing 3% BSA supplemented with Tween® 20. After blocking, the primary antibody was incubated overnight at 4°C. For progesterone receptor (PR) staining, rabbit anti-PR antibody (400-fold dilution, Cat. No. ab101688, Abcam) was used, and for estrogen receptor (ER) staining, rabbit anti-ER antibody (100-fold dilution, Cat. No. ab75635, Abcam) was used. After staining, the tissue was incubated with Histofine Simple Stain MAX-PO (Nichirei) for 30 minutes at room temperature. After the incubation, color development was performed using 3,3'-diaminobenzidine, and counterstaining was performed using hematoxylin. After staining, pathological diagnosis of the uterine corpus was performed using an optical microscope (BZ-9000, Keyence). The results are shown in Figure 10.

Figure 10 shows photographs of immunohistochemical staining of uterine hormone receptors. In Figure 10, in each row of photographs, the upper photograph shows a stained image of the progesterone receptor (PR), and the lower photograph shows a stained image of the estrogen receptor (ER). In Figure 10, the scale bar indicates 100 μm. As shown in Figure 10, in the control, expression of both PR and ER was confirmed. On the other hand, in endometrial cancer, expression of ER was observed, but expression of PR was not observed. From the above, it was found that PR expression was lost in endometrial cancer obtained using the method disclosed herein.

In general, there are cases of human endometrial cancer in which PR and/or ER expression is no longer observed. Furthermore, PR-negative cancers are known to be less sensitive to progesterone hormone therapy. Based on the above, it has been suggested that the triple mutant endometrial cancer disclosed herein, in which loss-of-function mutations have been introduced into all target genes of Tp53, Pten, and Lkb1, is a model in which hormone therapy is not expected to be effective, just like human cancers.

The present disclosure has been described above with reference to embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. Various modifications that would be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

All references cited herein, including scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth.

<Additional Notes>
Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes.
<Method of producing a non-human uterine cancer model animal>
(Appendix 1)
a mutation introduction step of contacting genomic DNA of a non-human animal uterine epithelial cell with a nucleic acid editing module to introduce a loss-of-function mutation into a target gene,
The target gene comprises a PTEN gene and a liver kinase B1 (LKB1) gene.
(Appendix 2)
A manufacturing method described in Appendix 1, comprising an introduction step of introducing the nucleic acid editing module into the uterine epithelial cells from outside the uterine epithelial cells.
(Appendix 3)
The method of claim 2, wherein the introduction is carried out by electroporation.
(Appendix 4)
The nucleic acid editing module includes a first nucleic acid editing module and a second nucleic acid editing module;
the first nucleic acid editing module is capable of introducing a loss-of-function mutation into the PTEN gene;
4. The method of any one of claims 1 to 3, wherein the second nucleic acid editing module is capable of introducing a loss-of-function mutation into the LKB1 gene.
(Appendix 5)
The nucleic acid sequence editing module is a CRISPR-Cas system;
The CRISPR-Cas system
a guide strand comprising a nucleic acid sequence that specifically binds to the nucleic acid sequence of the target gene;
a Cas protein; and
Including,
The manufacturing method described in any one of appendix 1 to 4, wherein the guide strand and the Cas protein form a complex.
(Appendix 6)
the nucleic acid editing module is a nucleic acid encoding the nucleic acid editing module,
A manufacturing method described in any of Appendix 1 to 4, comprising an expression step of expressing the nucleic acid editing module encoded by the nucleic acid.
(Appendix 7)
The nucleic acid sequence editing module is a CRISPR-Cas system;
The CRISPR-Cas system
a guide strand comprising a nucleic acid sequence that specifically binds to the nucleic acid sequence of the target gene;
a Cas protein; and
The method of claim 6, comprising:
(Appendix 8)
The method of any one of appendices 1 to 7, wherein the target gene comprises the Tp53 gene.
(Appendix 9)
the nucleic acid editing module comprises a third nucleic acid editing module;
The manufacturing method described in Appendix 8, wherein the third nucleic acid editing module is capable of introducing a loss-of-function mutation into the Tp53 gene.
(Appendix 10)
the uterine epithelial cells are endometrial epithelial cells;
The method of any one of appendices 1 to 9, wherein the uterine cancer is endometrial cancer.
(Appendix 11)
The manufacturing method described in any one of appendices 1 to 10, wherein the nucleic acid editing module does not contain Cre recombinase.
(Appendix 12)
12. The method of any one of claims 1 to 11, wherein the genomic DNA of the uterine epithelial cells does not contain a loxP sequence.
(Appendix 13)
A manufacturing method described in any one of appendices 1 to 12, wherein the nucleic acid editing module does not include a viral vector.
(Appendix 14)
The method of any one of claims 1 to 13, wherein the uterine cancer is progesterone receptor negative.
(Appendix 15)
The method of any one of claims 1 to 14, wherein the uterine cancer is estrogen receptor positive.
<Non-human animal model of uterine cancer>
(Appendix 16)
A non-human animal model of uterine cancer, in which the genomic DNA of uterine epithelial cells contains loss-of-function variants of the PTEN gene, the LKB1 gene, and the Tp53 gene.
(Appendix 17)
17. The non-human animal of claim 16, wherein the genomic DNA of the uterine epithelial cells does not contain loxP sequences.
(Appendix 18)
the uterine epithelial cells are endometrial epithelial cells;
18. The non-human animal of claim 16 or 17, wherein the uterine cancer is endometrial cancer.
(Appendix 19)
19. A non-human animal according to any one of appendices 16 to 18, in which a tumor derived from the uterine epithelial cells is formed.
(Appendix 20)
20. The non-human animal of any of claims 16 to 19, wherein the uterine cancer is progesterone receptor negative.
(Appendix 21)
21. The non-human animal of any one of claims 16 to 20, wherein the uterine cancer is estrogen receptor positive.
<Method for evaluating test substances>
(Appendix 22)
an administration step of administering a test substance to a non-human animal model of uterine cancer;
and evaluating uterine cancer in the non-human uterine cancer model animal,
The method for evaluating a test substance, wherein the non-human animal model of uterine cancer is a non-human animal model of uterine cancer obtained by a method for producing a non-human animal model of uterine cancer described in any one of Appendices 1 to 15, and/or a non-human animal model of uterine cancer described in any one of Appendices 16 to 21.
(Appendix 23)
The evaluation method described in Appendix 22, wherein the evaluation step includes a selection step of selecting a test substance that suppresses the occurrence of uterine cancer, reduces the size of uterine cancer, and/or suppresses the growth of uterine cancer as a candidate therapeutic substance for uterine cancer.

As described above, according to the present disclosure, a non-human animal model of uterine cancer can be produced from a wild-type non-human animal. Furthermore, according to the present disclosure, a non-human animal model of uterine cancer having uterine epithelial cell-derived uterine cancer can be produced. Therefore, the present disclosure is extremely useful in, for example, the fields of life science and medicine.

Claims (13)

a mutation introduction step of contacting genomic DNA of a non-human animal uterine epithelial cell with a nucleic acid editing module to introduce a loss-of-function mutation into a target gene,
The target genes include the PTEN gene , the liver kinase B1 (LKB1) gene, and the Tp53 gene . The manufacturing method described in claim 1, which includes an introduction step of introducing the nucleic acid editing module into the uterine epithelial cells from outside the uterine epithelial cells. The manufacturing method described in claim 2, wherein the introduction is performed by electroporation. The nucleic acid editing module includes a first nucleic acid editing module and a second nucleic acid editing module;
the first nucleic acid editing module is capable of introducing a loss-of-function mutation into the PTEN gene;
The method of claim 1 , wherein the second nucleic acid editing module is capable of introducing a loss-of-function mutation into the LKB1 gene. The nucleic acid editing module is a CRISPR-Cas system,
The CRISPR-Cas system
a guide strand comprising a nucleic acid sequence that specifically binds to the nucleic acid sequence of the target gene;
a Cas protein; and
Including,
The method of claim 1 , wherein the guide strand and the Cas protein form a complex. the nucleic acid editing module is a nucleic acid encoding the nucleic acid editing module,
The manufacturing method according to any one of claims 1 to 4, comprising an expression step of expressing the nucleic acid editing module encoded by the nucleic acid. the nucleic acid editing module comprises a third nucleic acid editing module;
The manufacturing method of claim 6 , wherein the third nucleic acid editing module is capable of introducing a loss-of-function mutation into the Tp53 gene. the uterine epithelial cells are endometrial epithelial cells;
The method of claim 1 , wherein the uterine cancer is endometrial cancer. A non-human animal model of uterine cancer that is progesterone receptor negative , in which the genomic DNA of uterine epithelial cells contains loss-of-function variants of the PTEN gene, the LKB1 gene, and the Tp53 gene. The non-human animal of claim 9 , wherein the genomic DNA of the uterine epithelial cells does not contain loxP sequences. the uterine epithelial cells are endometrial epithelial cells;
The non-human animal of claim 9 or 10 , wherein the uterine cancer is endometrial cancer. an administration step of administering a test substance to a non-human animal model of uterine cancer;
and evaluating uterine cancer in the non-human uterine cancer model animal,
12. A method for evaluating a test substance, wherein the non-human animal model of uterine cancer is a non-human animal model of uterine cancer obtained by the method for producing a non-human animal model of uterine cancer according to any one of claims 1 to 8, and/or the non-human animal model of uterine cancer according to any one of claims 9 to 11 . The evaluation method according to claim 12, wherein the evaluation step includes a selection step of selecting a test substance that suppresses the occurrence of, reduces the size of, and/or suppresses the growth of, uterine cancer as a candidate therapeutic agent for uterine cancer .