Exxon humanized mouse

Abstract

Provided are: a donor vector having an exon-humanized gene in which only an exon base sequence of a mouse gene is replaced with a human exon base sequence; an ES cell in which an endogenous mouse gene is replaced with said donor vector; and a mouse produced using said ES cell.

Description

Technical Field

[0001] The present invention relates to a vector having a gene in which the intron of a mouse gene remains as a mouse and only the exon is replaced with a human nucleotide sequence, embryonic stem cells (hereinafter referred to as "ES cells") having the exon-humanized gene, and an exon-humanized mouse established using the same.

Background Art

[0002] In 1980, transgenic mice were produced by injecting a foreign gene isolated by Gordon et al. into fertilized eggs (Gordon et al. Proc. Natl. Acad. Sci. USA. 77: 7380-7384, 1980). Since then, in order to introduce and express a human gene in a mouse, genomic genes of isolated human genes (for example, Nagata et al. J. Biochem. 117: 169-175, 1995), mini-genes (for example, Khiallan et al. J. Biol. Chem. 266: 23373-23379, 1991), artificial bacterial chromosomes (for example, Nieelsen et al. J. Biol. Chem. 272: 29752-29758, 1997), etc. have been injected into mouse fertilized eggs to produce transgenic mice, and there are many examples.

[0003] In 1989, a method was developed that could target and disrupt specific genes by homologous recombination using mouse ES cells (Zijlstra et al. Nature 342:435 - 438, 1989; Schwartzberg et al. Science 246:799 - 803, 1989). Using this homologous recombination method, methods are known for knocking in cDNA of human genes (Zhao et al. Gene Cells 13:1257 - 1268, 2008; Liu et al. Lab. Invest. 97:395 - 408, 2017), mini - genes (Lewis et al. Matrix Biol 31:214 - 226, 2012), etc. into the mouse locus to cause expression. However, the expression level of the introduced human gene was not necessarily normal, with many drawbacks such as being high, low, or having different tissue - specificities of expression.

[0004] From the above, transgenic mice produced by the above - mentioned method can be used for pathological analysis as, for example, human disease models. However, for use in the development of therapeutic methods and verification of effectiveness, normal expression levels and expression patterns are required. Therefore, there are limitations as a model in this regard.

Prior Art Documents

Non - Patent Documents

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[0006] An object of the present invention is to provide a mouse in which only exons among mouse genes are humanized. More specifically, in order to obtain a normal expression pattern in terms of gene expression level and tissue specificity of expression, an object is to provide an exon - humanized mouse in which the introns of the gene remain the mouse nucleotide sequence and only the exons have the human nucleotide sequence. [Means for Solving the Problems]

[0007] The present inventor took the transthyretin (TTR) gene as a representative example to solve the above problems and conducted intensive research. As a result, mouse ES cells were prepared in which the introns of the mouse Ttr gene remained the mouse nucleotide sequence and only the exon part was replaced with the human nucleotide sequence. Through the production of chimeric mice, the inventors succeeded in producing a mouse in which only the exons of the Ttr gene were humanized. When analyzing the tissue specificity of the expression of the exon - humanized Ttr gene in this mouse, it was found that the expression pattern was the same as that of the mouse Ttr gene, and also that the amount of TTR in the blood was the same as the level of mouse TTR in wild - type mice, thus completing the present invention.

[0008] That is, the present invention is as follows. (1) A donor vector comprising a fragment containing the n exons of a target gene composed of n exons contained in the genome of a mouse, wherein the n exons are each replaced with an exon of a corresponding human target gene. (2) The donor vector according to (1), wherein the target gene is a transthyretin gene. (3) A vector that expresses a guide RNA for cleaving the genome immediately upstream of the first exon, the vector containing a DNA encoding a target sequence for cleavage, a tracrRNA, and a DNA cleaving enzyme. (4) A vector that expresses a guide RNA for cleaving the genome immediately downstream of the nth exon, the vector containing a DNA encoding a target sequence for cleavage, a tracrRNA, and a DNA cleaving enzyme. (5) Exon-humanized ES cells in which the intron portion has been replaced with the nucleotide sequence of a mouse gene and the exon portion has been replaced with the nucleotide sequence of a human gene, by introducing the donor vector according to (1) or (2) and the vectors according to (3) and (4) into ES cells. (6) An exon-humanized mouse produced using the ES cells according to (5). (7) A method for producing an exon-humanized mouse in which an exon of a mouse gene is replaced with an exon of a human gene, the method comprising the following steps: (a) A step of introducing the donor vector according to (1) or (2) and the vectors according to (3) and (4) into ES cells. (b) A step of creating a chimeric embryo from the ES cells obtained in step (a) and transplanting the chimeric embryo into a foster parent to create a chimeric mouse, and (c) A step of mating a male mouse among the chimeric mice obtained in step (b) with a female mouse to give birth to offspring mice. The method as described above. (8) A method for producing a disease model mouse, characterized by introducing a gene mutation involved in a disease into the exon of the exon-humanized mouse according to (6). An experimental animal for gene therapy, comprising the exon-humanized mouse described in (9)(6) or the disease model mouse produced by the method described in (8).

Effects of the Invention

[0009] According to the present invention, ES cells having a gene in which the intron part is replaced with the base sequence of a mouse gene and the exon part is replaced with the base sequence of a human gene are provided. The ES cells of the present invention can establish exon-humanized mice that express human TTR protein in a normal amount and with normal tissue specificity. The exon-humanized mice of the present invention have a normal expression pattern, both quantitatively and tissue-specifically, and are extremely useful for creating human disease model mice and observing the effects of drugs and gene therapy.

Brief Description of the Drawings

[0010]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0011] Hereinafter, the present invention will be described in detail. 1. Outline The present invention relates to ES cells in which the intron portion of a gene has been replaced with a gene in which the intron portion has a mouse nucleotide sequence and the exon portion has a human nucleotide sequence, and a mouse in which human proteins exhibit a normal expression pattern has been established from the ES cells. Generally, in transgenic mice into which human genes have been introduced, the human genes injected into fertilized eggs are integrated at random positions on chromosomes. Therefore, gene expression varies from individual to individual, and it is rare to show a normal expression pattern. In addition, even when cDNA or mini-genes are inserted into mouse genes, it results in changes in the genomic structure and it is rare to show a normal expression pattern.

[0012] Therefore, in the present invention, in order to establish a mouse in which human proteins show a normal expression pattern, a mouse in which only exons are humanized is established. To establish this exon-humanized mouse, a donor vector containing a gene with a mouse nucleotide sequence for introns and a human nucleotide sequence for exons was constructed, and ES cells in which it was replaced at the original mouse gene locus were successfully established. Using the ES cells, chimeric mice were produced, and production of germline chimeric mice transmitted to the germline was also successful.

[0013] The mouse of the present invention is a mouse in which only the exons of the mouse target gene are replaced with human exons. In a preferred embodiment of the present invention, the mouse of the present invention is a mouse in which mouse proteins are not expressed and only human proteins are expressed. In this embodiment, since only the introns have a mouse nucleotide sequence, the sequences involved in expression regulation remain mouse-derived sequences, and mouse transcription factors etc. bind and function normally. As a result, normal expression levels and tissue-specific expression patterns can be obtained.

[0014] 2. Preparation of donor vector In the production of the mouse of the present invention, first, a guide RNA prepared based on the CRISPR / Cas9 method and a donor vector with a mouse nucleotide sequence for introns and a human nucleotide sequence for exons are introduced into normal ES cells by electroporation, and homologous recombination occurs to replace the mouse endogenous gene with the donor vector, thereby creating ES cells in which exons in the mouse genome are replaced with human genes. In the present invention, the donor vector contains a fragment in which n exons of a target gene composed of n exons contained in the mouse genome are each replaced with an exon of the corresponding human target gene, where n represents the number of exons contained in the genome.

[0015] The upper panel of FIG. 1 shows a genomic map containing exons targeted for humanization on the genome and introns that are not targeted for humanization. FIG. 1 exemplifies the exons of the transthyretin gene, and the number of exons is 4 (number of exons: n = 4) (boxes indicated by numbers 1 to 4 in FIG. 1). Hereinafter, the present invention will be described by taking this transthyretin gene as an example. The mouse transthyretin gene is denoted as the "Ttr gene", and the human transthyretin gene is denoted as the "TTR gene".

[0016] There are several methods for creating a gene in which only the exons have the human nucleotide sequence and the introns have the mouse nucleotide sequence. For example, there is a method of cloning human exons and mouse introns respectively and finally ligating them. However, the restriction enzyme sites for ligation do not necessarily match, and the operation is difficult. As an efficient method, as shown in FIG. 1, a method is to synthesize DNA collectively for a human exon and the mouse intron portions upstream and downstream thereof and insert this into a vector. That is, in FIG. 1, the regions labeled "pUC57-Donor-Ex1-Ex2", "pUC57-Donor-Ex3", and "pUC57-Donor-Ex4" are synthesized by DNA, and the other regions (intron regions) are cloned. According to this method, the restriction enzyme sites for ligation can be freely selected. The intron portion can be easily cloned from the mouse genome by a well-known method. Finally, these DNA fragments may be ligated.

[0017] Among large-sized genes, there may be a number of exons that are difficult to accommodate in a single vector. In such a case, instead of using a single donor vector, it can also be prepared by dividing it into several donor vectors. That is, a total of n exons are divided into k subclasses (n1, n2, ··· n k individuals) (k represents the number of donor vectors), and in the same manner as above, donor vectors can be prepared for each exon of each subclass. For example, when there are 10 exons (n = 10), donor vectors are prepared for each exon of three subclasses (k = 3) having, for example, 4 exons (n1 = 4), 3 exons (n2 = 3), and 3 exons (n3 = 3).

[0018] 3. Preparation of guide RNA The expression vectors for the guide RNAs of the present invention are a vector that expresses a guide RNA for cleaving the genome immediately upstream of the first exon, and a vector that expresses a guide RNA for cleaving the genome immediately downstream of the nth exon (the fourth exon in the case of the mouse Ttr gene). Each vector contains a DNA encoding a target sequence for cleavage, a tracrRNA, and a DNA cleavage enzyme. "Immediately upstream" means 1 to 20 bases upstream of the 5' end of the first exon, and "immediately downstream" means 1 to 20 bases downstream of the 3' end of the nth exon. When n exons are divided into n1, n2, ··· n k subclasses, it is defined in the same manner as when not divided into subclasses. For example, in each subclass, it is defined as 1 to 20 bases upstream of the 5' end of the first exon, or 1 to 20 bases downstream of the 3' end of the n1, n2, ··· n k th exons.

[0019] The CRISPR / Cas9 system is a defense mechanism by which bacteria and the like selectively destroy the DNA of invading viruses and plasmids. Basically, the expression of this mechanism requires molecules such as crRNA, tracrRNA, and Cas9 with double-stranded DNA cleavage activity. There is a sequence in crRNA that binds complementarily to the DNA of viruses and the like, and it binds to tracrRNA in another part. tracrRNA binds to Cas9, and as a result, Cas9 is carried to the DNA site of the virus to which crRNA has bound. Then, Cas9 destroys the virus by cleaving the viral DNA at that site (Jinek et al. Science 337:816 - 821, 2012). It has been revealed that if these three are expressed, DNA can be cleaved at a specific sequence part even in mammalian cells, that is, genes can be destroyed (Ran et al. Nat Protoc. 8:2281 - 2308, 2013). Furthermore, vectors (e.g., pX330) that can express these three in one vector have also been developed (Sakuma et al. Sci.Rep. 4:5400, 2014).

[0020] A homepage that can search for which base sequences in a gene are most easily destroyed has also been published. Currently, for example, using CCTop - CRISPR / Cas9 target online predictor (https: / / crispr.cos.uni - heidelberg.de), if you search for the base sequence near the one you want to destroy, it shows a candidate 20bp sequence, and at the same time, it also shows places with similar sequences, so - called off - target sites. From among these, about three places can be selected in order of priority, incorporated into pX330, and the efficiency of double - stranded DNA cleavage can be examined using cultured cells (Mashiko et al. Sci.Report 3:3355, 2013).

[0021] 4. Isolation of knock - in ES cells To establish exon - humanized mice, it is necessary to replace the endogenous gene with an exon - humanized gene at the ES cell stage, rather than in adult mice.

[0022] Therefore, in the present invention, in order to replace the genes endogenous to ES cells with exon humanized genes, the ordinary homologous recombination method or the CRISPR / Cas9 method can be used. In the CRISPR / Cas9 method, a specific base sequence can be specifically cleaved, and using this, knockout mice can be efficiently produced (Wang et al. Cell 153:910-918, 2013). At this time, it has been shown that the DNA repair system is induced in cells, homologous recombination also occurs at a high rate, and knock-in is also possible (Yang et al. Cell 154:1370-1379, 2013).

[0023] Examples of the culture medium for ES cells include GMEM medium (Glasgow’s Minimal Essential Medium), DMEM (Dulbecco's Modified Eagle Medium), RPMI1640 medium, etc. In the culture medium, KSR (Knockout Serum Replacement), fetal bovine serum (FBS), basic fibroblast growth factor (bFGF), β-mercaptoethanol, non-essential amino acids, glutamic acid, sodium pyruvate, and antibiotics (such as penicillin, streptomycin, etc.) can be selected and appropriately added. After culturing ES cells for a predetermined period, the ES cells are recovered by incubating them in a medium containing EDTA or collagenase IV. The recovered ES cells can also be passaged multiple times by culturing them in the presence or absence of feeder cells as necessary. Incidentally, the culture of inner cell mass under feeder-free conditions can be carried out in a medium conditioned by MEF.

[0024] The cultured ES cells can generally be identified using their marker genes. Examples of the marker genes for ES cells include Oct3 / 4, alkaline phosphatase, Sox2, Nanog, GDF3, REX1, FGF4, etc. The presence of the marker gene or gene product may be detected by any method such as PCR or Western blotting.

[0025] The substitution from the mouse gene to the exon-humanized gene can be carried out according to the CRISPR / Cas9 method. First, the above donor vector and guide RNA are introduced into ES cells by electroporation.

[0026] When the donor vector and guide RNA are introduced into ES cells, the specific sequence contained in the guide RNA binds to the complementary sequence on the genome, and double-strand cleavage of DNA occurs at that site by Cas9. As a result, a so-called homology directed repair system is induced, and homologous recombination occurs between the mouse base sequence contained in the donor vector and the homologous sequence on the mouse genome, and the exon-humanized gene is inserted (Figure 7).

[0027] According to this method, the endogenous mouse gene can be replaced with the exon-humanized gene. Figure 7 shows a diagram of the substituted allele. Here, in Figure 7, the numbers 1 to 4 in the diagram of "Ttr wild allele" represent exons 1 to 4 of the mouse transthyretin gene, and the numbers 1 to 4 in the diagram of "Humanized allele" represent exons 1 to 4 of humans.

[0028] The same applies to genes other than the Ttr gene. That is, a gene having a human base sequence in the exon part and a mouse base sequence in the intron part is prepared, and this gene and the guide RNA are introduced into ES cells by the CRISPR / Cas9 method.

[0029] For example, when producing a mouse in which only the exon of the Rbp4 gene is humanized, the exon-humanized Rbp4 hRBP4exon gene having a human base sequence in the four exons and a mouse base sequence in the intron is prepared, and this gene is introduced into ES cells together with the guide RNA, then hRBP4exon a mouse can be produced.

[0030] 5. Production of chimeric mice The production of chimeric mice can be carried out by a standard method. First, the established ES cells are aggregated with 8-cell stage embryos or injected into the blastoderm method. The embryo thus produced is called a chimeric embryo, and a chimeric mouse is produced by transplanting this chimeric embryo into the uterus of a pseudopregnant foster mother and giving birth. For example, to produce a chimeric embryo, first, a female mouse that has been superovulated with a hormonal agent is mated with a male mouse. Then, after a predetermined number of days, the early developing embryo is recovered from the oviduct or uterus. ES cells are aggregated or injected into the recovered embryo to produce a chimeric embryo.

[0031] Here, the "embryo" means an individual in the stage from fertilization to birth in ontogeny, including 2-cell stage embryos, 4-cell stage embryos, 8-cell stage embryos, morula embryos, blastocysts, etc. When using an 8-cell stage embryo, the early developing embryo can be recovered from the oviduct or uterus on the 2.5th day after fertilization, and when using a blastocyst, it can be recovered on the 3.5th day after fertilization. As a method for producing an aggregate using ES cells and an embryo, known techniques such as the microinjection method and the aggregation method can be used. The "aggregate" means an aggregate formed by ES cells and an embryo gathering in the same space, meaning either a form in which ES cells are injected into the embryo or a form in which the embryo is broken into individual cells and aggregated together with ES cells.

[0032] When adopting the microinjection method, ES cells are injected into the recovered embryo to produce a cell aggregate. When adopting the aggregation method, ES cells can be sprinkled on a normal embryo from which the zona pellucida has been removed and aggregated. On the other hand, a pseudopregnant female mouse for use as a foster mother can be obtained by mating a female mouse with a normal estrous cycle with a castrated male mouse by vasectomy or the like. A chimeric mouse can be produced by transplanting the chimeric embryo produced by the above method into the uterus of the produced pseudopregnant mouse and then giving birth.

[0033] 6. Production of exon humanized mice From among such chimeric mice, male mice derived from ES cell-transplanted embryos are selected. After the selected male chimeric mice mature, these mice are mated with female mice of an inbred mouse strain. Then, in the born offspring mice, it can be confirmed that the ES cells have been introduced into the germline of the chimeric mice by the appearance of the coat color of the mouse derived from the ES cells.

[0034] Whether the born offspring mice have the exon-humanized gene and whether they have a normal sequence can be identified by cutting the DNA with a restriction enzyme and detecting whether a DNA fragment of the target size is detected, and by analyzing the nucleotide sequence of the DNA. Once it can be confirmed that they have a normal sequence, in subsequent generations, exon-humanized mice can be identified by PCR analysis using the sequences of the human exon and the mouse intron as primers.

[0035] 7. Evaluation of Exon-Humanized Mice The fact that the exon has been humanized can be confirmed by measuring transthyretin in the serum by ELISA or Western blotting, distinguishing it from mouse transthyretin. When other genes are humanized, the expression proteins of the other genes can also be confirmed by ELISA or Western blotting.

[0036] 8. Generation of Mutant Exon-Humanized Mice Using Exon-Humanized Mice There are numerous human genetic diseases caused by a single gene abnormality. For example, familial amyloid polyneuropathy, an autosomal dominant disease, is caused by a point mutation in the TTR gene. By injecting crRNA, tracrRNA, Cas9, and a donor oligo into the fertilized eggs of mice with wild-type human TTR exons (wild-type exon humanized mice), the GTG encoding valine at position 30 can be replaced with the sequence of ATG (Yang et al. Cell 154:1370-1379, 2013). Thus, if the exon of the mouse gene is replaced with the human wild-type exon sequence to produce a wild-type exon humanized mouse, and then the human exon in the mouse is mutated, a mouse with a mutant exon, that is, a disease model mouse with the same mutant gene as the human patient, can be produced.

[0037] 9. Gene therapy experiments using wild-type exon humanized and mutant exon humanized mice By mating wild-type exon humanized and mutant exon humanized mice, heterozygous mice with wild-type and mutant genes can be obtained. The genotype of this mouse is the same as that of the human patient. Using this exon humanized heterozygous mouse, it can be used to verify treatment methods. In particular, gene therapy experiments for genetic diseases caused by abnormalities in genes expressed in the liver, which have attracted attention in recent years, can be conducted. Previously, mutations were induced in mouse genes and treatment experiments were conducted using them as models (Yin et al. Nat Biotechnol. 32:551-553, 2014; Pankowicz et al. Nat Commun .7:12642,2016; Yang et al. Nat Biotechnol 34:334 - 338,2016; Yin et al. Nat Biotechnol. 34:328 - 333,2016; Jarrett et al. Sci Rep. 7:44624,2017; Villiger et al. Nat Med. 24:1519 - 1525,2018). However, the nucleotide sequences are not the same as those of human genes, and when actually applied to humans, although they can be used as a reference, the therapeutic effects cannot necessarily be accurately predicted. In exon - humanized mice, since the exons have the same sequences as those in humans and the therapeutic effects can be reliably predicted, their usefulness is high. Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples. Regarding these experiments, etc., applications were made to the Animal Experiment Committee and the Recombinant DNA Experiment Safety Committee of the Second Type, and all were approved.

Example

[0038] Donor vector Donor vectors with the coding region sequences replaced from mouse to human for each of the four exons were prepared by the following method (Figure 1). Since Exon 1 and Exon 2 are in the vicinity, they were synthesized as one donor DNA (pUC57-DonorEx1-Ex2). This contains the mouse nucleotide sequence from the AvrII site to just before the ATG, the human nucleotide sequence of exon 1 starting from the ATG, the mouse nucleotide sequence of intron 1, the human nucleotide sequence of exon 2, and the mouse nucleotide sequence of intron 2 from the splice donor to the SacI site. The donor DNA of Exon 3 (pUC57-Donor-Ex3) was synthesized with the mouse nucleotide sequence from the XbaI site of mouse intron 2 to the splice acceptor, the human nucleotide sequence of exon 3, and the mouse nucleotide sequence of intron 3 from the splice donor to BclI. The donor DNA of Exon 4 (pUC57-Donorr-Ex4) was synthesized with the mouse nucleotide sequence from the SspI site of intron 3 to the splice acceptor, the human nucleotide sequence of exon 4, and the mouse nucleotide sequence from after the stop codon of the 3’ non-coding region to the EcoRI site. In addition, the 5’ homologous arm (2.8 kb), intron 2 (3.4 kb), intron 3 (3.5 kb), and 3’ homologous arm (2.9 kb) were amplified as DNA fragments using the genomic DNA of the ES cell RENKA strain of the C57BL / 6 line as a template. The above seven DNA fragments were ligated to prepare a donor vector (pBSK-TTR-all-in-one donor vector) (SEQ ID NO: 1) In the nucleotide sequence shown in SEQ ID NO: 1, the relationship between the sequences constituting the donor vector (pBSK-TTR-all-in-one donor vector) and the SEQ ID NO is shown in Table 1.

Table 1

[0039] To confirm the structure of the donor vectors we created, we digested them with restriction enzymes and analyzed the electrophoretic patterns. The observed DNA cleavage patterns were consistent with those predicted from the vector design (Figure 2). In addition, we determined the base sequences of the entire DNA amplified by each PCR and submitted them to the NCBJ. When the genomic sequence of the mouse was compared with that of a registered C57BL / 6J mouse, it was found to be identical, proving that the above method can be used to create genes in which only the exons are humanized.

[0040] Guide RNA The base sequences required for the production of guide RNA have already been reported in detail (Ran et al. Nat. Protoc. 8: 2281-2308, 2013). In addition, vectors (e.g., pX330) that can express crRNA, tracrRNA, and Cas9 in a single vector have been developed (Sakuma et al. Sci. Rep. 4: 5400, 2014), and are available from Addgene and other sources.

[0041] Based on the above papers, we analyzed the 20bp base sequence that can efficiently destroy exon 1 using search software (Crispr design tool). When selecting guide RNAs, we selected those with high scores and many mismatches so that the coding region after humanization would not be cut by gRNA. As a result, we selected the following two candidate sequences (Figure 3). TIFF0007699749000009.tif15115

[0042] To evaluate the efficiency of the guide RNAs into which these two arrays were inserted, the method of Mashiko et al. was utilized (Mashiko et al. Sci. Rep. 3:3355, 2013). First, each fragment of approximately 500 bp of the Ttr genome containing the target sequences of Cas9 (SEQ ID NOs: 15, 16) was inserted between the 5' and 3' sequences of the DasherGFP gene (DNA2.0 lnc.) downstream of the CMV promoter, and DasherGXXFP expression vectors (pDGXXFP-ex1, pDGXXFP-ex4) that do not emit fluorescence were constructed. These were introduced into HEK293T cells together with guide RNA-Cas9 expression vectors (pX330-Ex1-gRNA1, pX330-Ex1-gRNA2, pX330-Ex4-gRNA1, pX330-Ex4-gRNA2) targeting the corresponding regions. When the Cas9 protein guided by the gRNA cleaves the target sequence and the resulting ends are repaired by homology-directed repair, the base sequence of DasherGXXFP is reconstructed into DasherGFP, and the expressed protein emits fluorescence. The one determined to have a higher ratio of the number of fluorescent cells and higher cleavage activity of Cas9 was pX330-Ex1-gRNA2 (Figure 4).

[0043] Regarding exon 4, a search was conducted in the same manner, and the following two candidate sequences were selected (Figure 5). TIFF0007699749000010.tif15116 Note that the target sequences of Cas9 are shown in SEQ ID NOs: 19, 20 (Figure 5).

[0044] The efficiency of the guide RNAs into which these two arrays were inserted was evaluated in the same manner. The results are shown in Figure 6. The one determined to have a higher ratio of the number of fluorescent cells and higher cleavage activity of Cas9 was pX330-Ex4-gRNA2 (Figure 6).

[0045] Establishment of ES cells In this example, donor vectors and guide RNAs (pX330-Ex1-gRNA2 and pX330-Ex4-gRNA2) were used to establish ES cells for the establishment of exon-humanized mice. Donor DNA, vector (pBSK-TTR-all-in-one donor vector), and puro expression vector were co-introduced into ES cells (RENKA strain derived from C57BL / 6N strain) by electroporation. As a result, 10 clones in which all four exons were replaced with human TTR gene exons were obtained (Table 2). However, only 3 clones (a6221, a6226, a6232) showed the DNA fragments expected by restriction enzyme digestion, and unexpected mutations and deletions occurred in the others.

[0046] The nucleotide sequences of the above 3 clones were analyzed. As a result, in addition to the allele in which all exons were replaced with the human TTR gene, a6221 had alleles in which only exon 1, exon 2, and exon 4 were replaced with the human TTR gene, an allele in which all exons were replaced with the human TTR gene but only 1 base in exon 2 was not humanized, and an allele in which all exons were replaced with the human TTR gene but had a 1-base mutation in intron 1, so it was a so-called mosaic. In addition to the allele in which all exons were replaced with the human TTR gene, a6226 had an allele in which all exons were replaced with the human TTR gene but had a 1-base mutation in intron 2, and it was also a mosaic. a6232 had only the allele in which all exons were replaced with the human TTR gene. In addition, no wild-type allele was observed in these ES cell clones. From the above results, it was revealed that a6221, a6226, and a6232 all had alleles in which all exons were replaced with the exons of the human TTR gene.

Table 2

[0047] Mouse strain (TtrhTTRexon ) Establishment Using the above homologous recombinant ES cell clones (a6221, a6226, a6232) and 8-cell stage embryos of the ICR strain, chimeric embryos were produced by the aggregation method. On the expected date of parturition of the recipient female mice transplanted with these chimeric embryos, parturition was confirmed, and pregnant mice that had not given birth were subjected to cesarean section. The obtained chimeric mice were reared until weaning, and the chimerism rate was determined by coat color at the time of weaning. The chimerism rate was visually determined based on the coat color rate of the whole body. Five 100% chimeric mice were obtained from ES cells derived from a6221, but the chimerism rate was low for the others (Table 3).

Table 3

[0048] F1 progeny were produced by mating the 100% chimeric mice obtained from the above a6211 with wild-type mice. As a result, ES-derived individuals were obtained from all chimeras. After DNA extraction from the body tissues of the F1 progeny, the humanized exons were detected under PCR conditions using primers specific to the humanized alleles (Figure 7). As a result, for exon1, exon2, and exon4, the exons were humanized in all individuals, and for exon3, it was confirmed that the exons were humanized in 16 individuals (8 males and 8 females) (Figure 8). Next, for each exon of the 16 individuals (No. F1-1, F1-3, F1-5, F1-6, F1-7, F1-8, F1-9, F1-12, F1-14, F1-15, F1-19, F1-20, F1-25, F1-28, F1-30, F1-33) in which all exons were humanized, the nucleotide sequences were analyzed by direct sequencing to confirm that there were no unexpected mutations in the humanized sequences.

[0049] First, a 2.9 kbp DNA region containing exon1 and exon2 was amplified using the primers shown in Figure 9 above under the PCR conditions shown on the left in Figure 9. TIFF0007699749000013.tif29124

[0050] A 2.9-kb band was detected in all cases (right panel of Fig. 9). These PCR products were digested with BamHI to digest the mouse sequences (1.8 kbp, 1.1 kbp) (lower right panel of Fig. 9). After purifying the humanized sequence (2.9 kbp) that was not cleaved by BamHI, direct sequencing was performed. As a result, for the 16 humanized individuals, it was confirmed that there were no differences from the expected sequences in both exon 1 (Fig. 10) and exon 2 (Fig. 11). In Fig. 10, the nucleotide sequence of mouse exon 1 is shown as SEQ ID NO: 25, and the nucleotide sequence of human exon 1 is shown as SEQ ID NO: 26. In Fig. 11, the nucleotide sequence of mouse exon 2 is shown as SEQ ID NO: 27, and the nucleotide sequence of human exon 2 is shown as SEQ ID NO: 28.

[0051] In addition, the mutation in intron 1 confirmed in the ES cell clone (a6221) was not observed. Therefore, this mutation is considered to be an error during PCR amplification. Similarly, a 1096-bp DNA region containing exon 3 was amplified using the primers shown above in Fig. 12 (below) under the PCR conditions shown on the left in Fig. 12. TIFF0007699749000014.tif21123

[0052] These PCR products were digested with EcoRI to digest the mouse sequences (659 bp, 437 bp) (lower right panel of Fig. 12). After purifying the humanized sequence (1096 bp) that was not cleaved by EcoRI, direct sequencing was performed. As a result, for the 16 individuals in which all exons were humanized, it was confirmed that there were no differences from the expected sequences (Fig. 13). In Fig. 13, the nucleotide sequence of mouse exon 3 is shown as SEQ ID NO: 32, and the nucleotide sequence of human exon 3 is shown as SEQ ID NO: 33.

[0053] Furthermore, a 1543-bp DNA region containing exon 4 was amplified using the primers shown above in Fig. 14 (below) under the PCR conditions shown on the left in Fig. 14 (right panel of Fig. 14). TIFF0007699749000015.tif21119

[0054] These PCR products were treated with MscI to digest the mouse sequences (863 bp, 680 bp) (lower right in Figure 14). After purifying the humanized sequences (1543 bp) that were not cleaved by MscI, direct sequencing was performed. As a result, for 16 individuals in which all exons were humanized, it was confirmed that there were no differences from the expected sequences (Figure 15). In Figure 15, the nucleotide sequence of mouse exon 4 is shown as SEQ ID NO: 37, and the nucleotide sequence of human exon 4 is shown as SEQ ID NO: 38.

[0055] In addition, to analyze the presence or absence of large genomes that cannot be analyzed by sequencing alone, 16 individuals in which all exons were humanized were analyzed by Southern hybridization. By this analysis, for any restriction enzyme, only the expected DNA fragments (11.4 kb for the 5’ probe and 8.6 kb for the 3’ side) were detected on both the 5’ side (Figure 16) and the 3’ side (Figure 17). From the above results, 16 (8 males and 8 females) F1 mice were determined to be heterozygous mice. The analysis results are summarized in Table 4.

Table 4

[0056] Evaluation of exon humanized mice (Ttr hTTRexon ) The expression of the exon humanized gene can be confirmed by examining the following items alone or in appropriate combinations.

[0057] The tissue specificity of the expression of the exon humanized gene was analyzed by Northern blotting using RNA extracted from each organ and tissue. Wild-type mice (Ttr + / + ) and TTR gene exon humanized mice (Ttr hTTRexon / hTTRexonFor [[ID=]], brains, eyeballs, hearts, lungs, livers, kidneys, spleens, and skeletal muscles were excised from 12-week-old mice after birth, and RNA was extracted. Total RNA was prepared to be 2 μg or 10 μg. An equal volume of NorthernMax (registered trademark)-Gly Sample Loading Dye (Thermo Fisher Scientific Inc., #AM8551) was added thereto and mixed, and electrophoresis was performed using a 1% formalin-denaturing gel. After electrophoresis, blotting was performed onto a nylon membrane by the capillary method using 20×SSC. After blotting, the membrane was air-dried, and RNA was fixed onto the membrane using a UV crosslinker. Of this membrane, the one using 2 μg of RNA was used for the beta-actin probe, and the one using 10 μg of RNA was used for the Ttr probe.

[0058] To detect mouse Ttr of wild-type mice and human TTR of exon-humanized mice of the TTR gene by Northern blot, a Ttr probe was designed for the 5’UTR, which is a sequence common to both (upper left in Figure 18). As a probe for detecting the Ttr gene, primers Ttr-5’UTR-F1 (5’-CTA ATC TCC CTA GGC AAG GTT CAT A-3’ (SEQ ID NO: 39)) and Ttr-5’UTR-R2 (5’-AAG CCA TCC TGT CAG GAG CTT GTG G-3’ (SEQ ID NO: 40)) were used, PCR was performed using cDNA of wild-type mice as a template, and a purified 196-bp fragment amplified thereby was prepared. After fragment purification, the probe was labeled according to the protocol attached to the kit using the AlkPhos Direct Labelling and Detection System (GE Healthcare). Using this and the membrane obtained by blotting the 10 μg of RNA extracted from the liver of wild-type mice after electrophoresis and the labeled Ttr probe, hybridization was performed at 55°C for 18 hours. As a result, Ttr hTTRexon / hTTRexon and Ttr + / + showed specific signals in the brains, eyes, and livers (middle in Figure 18). The expression pattern of the exon-humanized gene was also the same as that of the mouse endogenous Ttr gene.

[0059] As an internal control, hybridization was performed using beta-actin as a probe. The position of the beta-actin probe is shown in the upper right of Figure 18. Primers 5'-GGT CAG AAG GAC TCC TAT GTG GG-3' (SEQ ID NO: 41) and 5'-ATG AGG TAG TCT GTC AGG TC-3' (SEQ ID NO: 42) were used to amplify by PCR using mouse liver cDNA as a template, and this fragment was cloned into pBluescriptSK. The probe DNA excised from the plasmid with a restriction enzyme was labeled as a probe by the same procedure as for the Ttr probe preparation, and hybridization was performed at 55°C for 18 hours. As a result, it was detected in a similar pattern in exon-humanized mice and wild-type mice (lower part of Figure 18).

[0060] TTR protein is produced in the liver and secreted into the blood. Therefore, by measuring TTR in the blood, the expression level can be accurately analyzed. The ELISA (Enzyme-Linked Immuno Sorbent Assay) method or Western blot method is used, and in the case of human TTR, it was measured using a commercially available ELISA measurement kit. The concentration measurement of human TTR by ELISA was performed using the following TTR measurement ELISA kit (Human Prealbumin (Transthyretin, TTR) ELSIA kit, manufacturer model number: AssayPro·EP3010·1) to measure the human TTR concentration in the sera of 5 male and 5 female mice at 12 weeks of age. hTTRexon / hTTRexon It was measured using the sera of 5 male and 5 female mice. The measured concentration of each sample was determined from the calibration curve (31.25, 7.81, 1.95, 0.49, 0.12, 0 ng / ml) obtained using the standard product attached to the kit. The serum concentration of each sample was corrected and calculated from the measured concentration and the dilution ratio. As a result, it was 152.42 ± 8.97 μg / ml in females and 185.72 ± 14.79 μg / ml in males of Ttr (left part of Figure 19). hTTRexon / hTTRexon

[0061] ​Mouse TTR was measured by Western blotting. Serum from wild-type mouse Ttr at 12 weeks of age was used to measure mouse TTR concentration by Western blot analysis. As the mouse TTR detection antibody, an anti-TTR antibody (Proteintech, 11891-1-AP) was used, and recombinant mouse TTR (mTTR: LifeSpan BioSciences, LS-G12719) was used for calibration curve preparation. As a result, in female Ttr, it was 137.93 ± 4.37 μg / ml, and in male, it was 136.27 ± 4.59 μg / ml (right in Figure 19). + / + Using the serum of + / + , mouse TTR concentration was measured by Western blot analysis. As the mouse TTR detection antibody, an anti-TTR antibody (Proteintech, 11891-1-AP) was used, and for calibration curve preparation, recombinant mouse TTR (mTTR: LifeSpan BioSciences, LS-G12719) was used. As a result, in female Ttr + / + it was 137.93 ± 4.37 μg / ml, and in male, it was 136.27 ± 4.59 μg / ml (right in Figure 19). From the above, it was found that the amount of human TTR of Ttr hTTRexon / hTTRexon and the amount of mouse TTR of

Example

[0062] Production of homozygous mice (Ttr + / hV30exon ) from wild-type exon humanized heterozygous mice (Ttr hV30exon / hV30exon ) The established wild-type exon humanized heterozygous mice (Ttr + / hV30exon ) were mated to obtain a large number of wild-type exon humanized homozygous mice (Ttr hV30exon / hV30exon ).

[0063] Ttr hV30exon / hV30exon Production of mutant exon humanized heterozygous mice (Ttr hV30exon / hM30exon ) from By in vitro fertilization using eggs obtained by superovulation of female Ttr hV30exon / hV30exon and sperm of male Ttr hV30exon / hV30exo , a large number of Ttr hV30exon / hV30exonFertilized eggs are obtained. CrRNA, tracrRNA, Cas9 mRNA, and single-strand DNA (ssDNA) are introduced into the fertilized eggs by the already established electroporation method (Figure 20). The sequences of crRNA, tracrRNA, and ssDNA are shown in Figure 21 (SEQ ID NOs: 43-47). In the fertilized eggs after injection, homologous recombination occurs between the donor oligo and the human TTR gene on the genome, and the 30th GTG is replaced by ATG. When it reaches the 2-cell stage embryo, it is transplanted into the oviduct of the foster mother to obtain offspring. Genotyping of the born mice is performed, and Ttr mice heterozygous for wild-type and mutant exons are selected. hV30exon / hM30exon Select Ttr mice.

[0064] Ttr hV30exon / hM30exon Ttr from mice hM30exon / hM30exon and Ttr hV30exon / hM30exon production increase The Ttr mice obtained above hV30exon / hM30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below. hV30exon / hM30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below. hV30exon / hM30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below. hM30exon / hM30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below. hV30exon / hV30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below. hM30exon / hM30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below. hV30exon / hM30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below. hV30exon / hM30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below. hV30exon / hM30exon Just mating the Ttr mice only results in only 1 / 2 of them being Ttr mice, so the efficiency is low. Therefore, first, a large number of Ttr mice are obtained by mating Ttr mice. Then, in vitro fertilization is performed using the eggs and sperm of Ttr and Ttr, and all of them will have the genotype of Ttr. Thus, Ttr mice can be mass-produced. Ttr mice have the same genotype as human patients and can be used in the gene therapy experiments described below.

Example

[0065] Simultaneous disruption of human wild-type TTR gene (TTRVal30) and human mutant TTR gene (TTRMet30) Ttr hV30exon / hM30exon Using Ttr mice, experiments can be performed to disrupt both wild-type and mutant genes, and experiments to disrupt only the mutant gene. First, the former will be described. Ttr hV30exon / hM30exonTo disrupt the TTR gene in the liver, the CRISPR / Cas9 method is used. To completely suppress gene expression, it is most certain to disrupt the ATG, which is the translation initiation codon. For this purpose, a target sequence was searched using a website on the Internet called CCTop (https: / / crispr.cos.uni-heidelberg.de). Since it is said that double-strand cleavage occurs 3 bp upstream of the PAM sequence (Jinek et al. Science 337:816-821, 2012), TCCACTCATTCTTGGCAGG containing ATG A(TG G) (SEQ ID NO: 48: The TGG at the right end is the PAM sequence. The underlined part is ATG) was the best sequence.

[0066] This cleavage activity can be evaluated by the method of Mashiko et al. already described (Mashiko et al. Sci. Rep. 3:3355, 2013). A gene fragment of about 0.5 kb - 1.0 kb containing the target sequence near the center is introduced into the multiple cloning site of the plasmid pCAG-EGxxFP (obtained from addgene). pCAG-EGxxFP is a sequence on the N-terminal side and the C-terminal side with an overlapping sequence of about 500 bp for the EGFP gene, and has a structure that sandwiches the target sequence (0.5 - 1.0 kb). The pCAG-EGxxFP into which the target sequence has been introduced and the pX330 into which the guide RNA sequence has been introduced are co-introduced into HEK293 cells. In HEK293 cells, gRNA and CAS9 are expressed, bind to the target sequence introduced into pCAG-EGxxFP, and double-strand cleavage occurs. Then, the overlapping part of the EGFP on the N-terminal side and the EGFP sequence on the C-terminal side undergoes gene homologous recombination or single-strand annealing, recombines to form a complete EGFP sequence, EGFP is expressed, and green fluorescence is emitted. That is, by comparing the number of cells emitting green fluorescence, the cleavage activity for each target sequence can be relatively evaluated, and the most efficient target sequence can be determined. The above sequence was incorporated into pX330 to prepare pX330-ATG (Figure 22). In Figure 22, the target sequence of Cas9 is shown in SEQ ID NO: 49, and the sequence with the "cacc" sequence added to the 5' side and the "caaa" sequence added to the 3' side of the target sequence is shown in SEQ ID NO: 50.

[0067] Delivery of pX330-ATG to the liver Ttr around 6 weeks of age hV30eon / hM30exon 2 ml of a solution containing 50 μg of pX330-ATG (1 / 10 of the mouse's body weight, 20 g) is injected into the tail vein of the mouse within 5-7 seconds (hydrodynamic method: Lewis et al. Nat. Genet. 32: 107-108, 2002). With this method, pX330-ATG can be introduced into up to 80% of the hepatocytes.

[0068] Evaluation of TTR gene disruption The extent of TTR gene disruption in the liver can be assessed by the following methods. (1) Liver DNA analysis: At about 8 weeks of age, a partial hepatectomy is performed. DNA is extracted and the base sequence near the ATG is analyzed to determine the frequency of remaining wild-type TTR, mutant TTR, and insertion and deletion mutations. (2) Serum TTR concentration: At the ages of 3, 6, 12, 18, and 24 months, human TTR in serum was measured by ELISA. The level of TTR in serum was also estimated by measuring the blood concentration, and the level of TTR gene destruction was compared with the DNA analysis data. (3) Analysis of non-fibrillar TTR deposits: Non-fibrillar TTR deposits can be seen as early as one month of age, preceding amyloid deposition, and amyloid deposition begins as early as one year after birth. Therefore, autopsies will be performed at 3, 6, 12, 18, and 24 months of age (targeting 10 animals per group), and the digestive tract, kidneys, heart, sciatic nerve, and spleen will be removed and fixed, and tissue sections will be prepared. The sections will be immunostained with anti-TTR antibodies and anti-serum amyloid A (SAA) antibodies. Using anti-SAA antibodies, amyloid deposition associated with inflammation, etc. can be analyzed and differential diagnosis can be made. In the future, red staining will also be performed to analyze amyloid deposition. (4) By integrating the above data, we will analyze the correlation between the rate of TTR gene destruction and blood TTR concentration and the amount of non-fibrous TTR, and clarify the possibility of treatment through gene destruction. EXAMPLES

[0069] Disruption of only the human mutant TTR gene (TTRMet30) Ttr hV30exon / hM30exon Using mice, it is possible to conduct an experiment to disrupt only the mutant gene. Ttr hV30exon / hM30exon To disrupt the TTR gene in the liver of Ttr, the CRISPR / Cas9 method is used. To disrupt only the mutant, only the gene having ATG encoding methionine, which is the 30th amino acid, is disrupted. For this purpose, the target sequence was searched using a website on the Internet called CCTop (https: / / crispr.cos.uni-heidelberg.de). Since double-strand cleavage is said to occur 3 bp upstream of the PAM sequence (Jinek et al. Science 337:816-821, 2012), TCCACTCATTCTTGGCAGG A(TG G) (SEQ ID NO: 48: The TGG at the right end is the PAM sequence. The underlined part is ATG) was the best sequence (Figure 23). In Figure 23, the target sequence of Cas9 is shown in SEQ ID NO: 51, and the sequence with the "cacc" sequence added to the 5' side and the "caaa" sequence added to the 3' side of the target sequence is shown in SEQ ID NO: 52. The cleavage activity can be evaluated by the method of Mashiko et al. described above (Mashiko et al. Sci. Rep. 3:3355, 2013).

[0070] Delivery of pX330-MET30 to the liver Ttr around 6 weeks of age hV30eon / hM30exon From the tail vein of Ttr around 6 weeks of age, 2 ml of a solution containing 50 μg of pX330-ATG (1 / 10 of the amount assuming a mouse body weight of 20 g) is injected within 5 - 7 seconds (hydrodynamic method: Lewin et al. Nat. Genet. 32:107-108, 2002). By this method, pS330-ATG can be introduced into up to 80% of hepatocytes.

[0071] Evaluation of TTR gene disruption The degree of disruption of the TTR gene in the liver can be evaluated by the following method. (1) Liver DNA analysis: At about 8 weeks of age, a partial hepatectomy is performed. DNA is extracted and the base sequence around the 30th ATG is analyzed to determine the frequency of remaining wild-type TTR, mutant TTR, and insertion and deletion mutations. (2) Serum TTR concentration: At the ages of 3, 6, 12, 18, and 24 months, human TTR in serum was measured by ELISA. The level of TTR in serum was also estimated by measuring the blood concentration, and the level of TTR gene destruction was compared with the DNA analysis data. (3) Analysis of non-fibrillar TTR deposits: Non-fibrillar TTR deposits can be seen as early as one month of age, preceding amyloid deposition, and amyloid deposition begins as early as one year after birth. Therefore, autopsies will be performed at 3, 6, 12, 18, and 24 months of age (targeting 10 animals per group), and the digestive tract, kidneys, heart, sciatic nerve, and spleen will be removed and fixed, and tissue sections will be prepared. The sections will be immunostained with anti-TTR antibodies and anti-serum amyloid A (SAA) antibodies. Using anti-SAA antibodies, amyloid deposition associated with inflammation, etc. can be analyzed and differential diagnosis can be made. In the future, red staining will also be performed to analyze amyloid deposition. (4) By integrating the above data, we will analyze the correlation between the rate of TTR gene destruction and blood TTR concentration and the amount of non-fibrous TTR, and clarify the possibility of treatment through gene destruction. [Industrial Applicability]

[0072] The present invention provides an exon-humanized TTR gene in which the exon of the Ttr gene is humanized, an ES cell into which the exon has been inserted, and an exon-humanized mouse derived from the ES cell. A mutation in the human TTR gene causes familial amyloid neuropathy, a dominant genetic disease. Using the mouse of the present invention, a mutation can be introduced into the human TTR gene to produce a human disease model mouse. Using this mouse, it becomes possible to perform a so-called gene therapy experiment in which the human TTR gene is destroyed in the liver, and non-clinical trials can be carried out to examine the therapeutic effect. [Sequence List Free Text]

[0073] SEQ ID NO: 1: Synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NOS: 12 - 24: Synthetic DNA SEQ ID NOS: 29 - 31: Synthetic DNA SEQ ID NOS: 34 - 36: Synthetic DNA SEQ ID NOS: 39 - 44: Synthetic DNA SEQ ID NO: 45: Synthetic DNA / RNA SEQ ID NO: 46: Synthetic RNA SEQ ID NOS: 47 - 52: Synthetic DNA [Sequence Listing] TIFF0007699749000017.tif251147TIFF0007699749000018.tif244148TIFF0007699749000019.tif244148TIFF0007699749000020.tif244148TIFF0007699749000021.tif245148TIFF0007699749000022.tif243148TIFF0007699749000023.tif243148TIFF0007699749000024.tif243148TIFF0007699749000025.tif244148TIFF0007699749000026.tif243147TIFF0007699749000027.tif243147TIFF0007699749000028.tif244148TIFF0007699749000029.tif244148TIFF0007699749000030.tif244148TIFF0007699749000031.tif244148TIFF0007699749000032.tif239148TIFF0007699749000033.tif245148TIFF0007699749000034.tif244148TIFF0007699749000035.tif244148TIFF0007699749000036.tif244147TIFF0007699749000037.tif245148TIFF0007699749000038.tif244148TIFF0007699749000039.tif250148TIFF0007699749000040.tif244148TIFF0007699749000041.tif244148TIFF0007699749000042.tif245148TIFF0007699749000043.tif250148TIFF0007699749000044.tif244148TIFF0007699749000045.tif249148TIFF0007699749000046.tif243148TIFF0007699749000047.tif244148TIFF0007699749000048.tif243148TIFF0007699749000049.tif243148TIFF0007699749000050.tif249148TIFF0007699749000051.tif231148TIFF0007699749000052.tif248148TIFF0007699749000053.tif249148TIFF0007699749000054.tif244148TIFF0007699749000055.tif248148TIFF0007699749000056.tif248148TIFF0007699749000057.tif239148TIFF0007699749000058.tif244148TIFF0007699749000059.tif249148TIFF0007699749000060.tif62148.

Claims

1. A donor vector for use in producing a human disease model mouse, comprising a fragment containing n exons of a target gene composed of n exons contained in the mouse genome, wherein the n exons are each replaced by an exon of a corresponding human target gene, and containing an intron containing the nucleotide sequence of the mouse gene. The target gene is the transthyretin gene.

2. A composition containing a vector for use together with the donor vector according to Claim 1, wherein the vector expresses a guide RNA for cleaving the genome immediately upstream of the first exon, and contains DNA encoding a target sequence for cleavage, tracrRNA, and a DNA cleaving enzyme.

3. A composition containing a vector for use together with the donor vector according to Claim 1, wherein the vector expresses a guide RNA for cleaving the genome immediately downstream of the nth exon, and contains DNA encoding a target sequence for cleavage, tracrRNA, and a DNA cleaving enzyme.

4. Exon-humanized ES cells in which the intron portion is replaced with the nucleotide sequence of the mouse gene and the exon portion is replaced with the nucleotide sequence of the human gene, by introducing the donor vector according to Claim 1 and the compositions according to Claims 2 and 3 into ES cells.

5. An exon-humanized mouse produced using the ES cells according to Claim 4.

6. A method for producing an exon-humanized mouse in which an exon of a mouse gene is replaced with an exon of a human gene, comprising the following steps: (a) A step of introducing the donor vector according to Claim 1 and the compositions according to Claims 2 and 3 into ES cells; (b) A step of producing a chimeric embryo from the ES cells obtained in step (a) and transplanting the chimeric embryo into a foster parent to produce a chimeric mouse; and (c) A step of mating male mice among the chimeric mice obtained in step (b) with female mice to give birth to offspring mice. The method as described above.

7. A method for producing a disease model mouse, characterized by introducing a gene mutation involved in a disease into an exon of the exon-humanized mouse according to Claim 5.

8. An experimental animal for gene therapy, comprising the exon-humanized mouse according to Claim 5 or a disease model mouse produced by the method according to Claim 7.

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