New Genomic Safe Harbors and Their Uses

Abstract

The present invention relates to a novel genomic safe harbor (GSH), a method for expressing a foreign gene using the same, and cells into which the foreign gene has been introduced by the method. The method for expressing a foreign gene using the novel genomic safe harbor of the present invention allows the foreign gene to be safely introduced without disrupting the transcription of adjacent genes, and allows the foreign gene to be expressed stably for a long period of time, thereby enabling the expression of a variety of foreign genes in cells without safety issues.

Description

[Technical Field]

[0001] The present invention relates to a novel genomic safe harbor (GSH) for gene insertion and a method for expressing a foreign gene using the same. Specifically, the genomic safe harbor of the present invention allows safe introduction of a foreign gene and stable expression of the foreign gene, thereby enabling the expression of various foreign genes in cells without safety issues.

[0002] [Background technology]

[0003] One of the most powerful tools for treating human diseases is the insertion of foreign genes (transgenes) into the human genome. Transgenes inserted into human cells can be used to treat diseases by altering the properties of the host cells, but various techniques must be applied to ensure precise gene delivery and sustained expression.

[0004] The most common method for inserting genes into the human genome is by using retroviral or lentiviral vectors. However, these vectors tend to insert near transcriptionally activated genes, which increases the likelihood of mutations in host cells. If mutations occur in cancer-related genes, disruption of their expression can lead to various types of cancer.

[0005] As an alternative to viral vectors, a site-specific gene insertion system based on CRISPR-Cas9 and homology-directed repair (HDR) is being used. CRISPR, an abbreviation for clustered regularly interspaced short palindromic repeats, is a genetic sequence that functions as a bacterial adaptive immune system. When bacteria are infected with a virus, the guide RNA (gRNA) expressed by CRISPR binds to the nuclease Cas9, recognizing and cleaving the portion of the viral genome that has a sequence complementary to the gRNA, thereby preventing infection. HDR is a process in which damaged DNA is repaired when a double-strand break (DSB) occurs using DNA fragments that share homology with the corresponding site sequence. By inducing a DSB in the genome using gRNA and Cas9 and simultaneously introducing a donor vector containing a homologous sequence to the foreign gene, precise gene insertion can be achieved.

[0006] Using technologies such as CRISPR-Cas9, researchers are exploring genomic regions suitable for introducing foreign genes. A region that maintains the expression of a foreign gene without disrupting the transcription of neighboring genes is called a genomic safe harbor (GSH). Previously studied GSH sites include the adeno-associated virus integration site 1 (AAVS1) on chromosome 19, but because it is located in a gene-dense region, it has the potential to disrupt the transcription of nearby genes (Sadelain et al., 2012), which tends to suppress the expression of introduced foreign genes (Ordovas et al., 2015). Therefore, new GSH candidates are needed to ensure the safe introduction and stable expression of foreign genes.

[0007] The present inventors have conducted extensive research to find regions suitable for new genomic safe harbors, and as a result, have completed the present invention by searching for new genomic safe harbor regions that do not disrupt the transcription of adjacent genes and that stably express foreign genes.

[0008] Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a novel genome safe harbor in which foreign genes can be safely introduced and stably expressed.

[0010] The present invention provides a method for expressing foreign genes in cells using a novel genomic safe harbor.

[0011] [Means for solving the problem]

[0012] The present invention provides a method for expressing an exogenous polynucleotide in a cell, comprising the steps of: introducing a polynucleotide encoding a nuclease into a cell and expressing the nuclease; allowing the nuclease to specifically bind to one or more nucleic acid regions selected from the group consisting of a first nucleic acid region from positions 24894446 to 24894525 on chromosome 9 of the human genome, a second nucleic acid region from positions 9064276 to 9064355 on chromosome 3, and a third nucleic acid region from positions 120174229 to 120174308 on chromosome 4, thereby cleaving the nucleic acid region; and introducing an exogenous polynucleotide into the cell and inserting it into the cleavage site of the nucleic acid region.

[0013] In one embodiment, the cells may be cells derived from human blood, body fluids, tissue, stem cells or carcinomas.

[0014] In one embodiment, the cell may be a somatic cell, a germ cell, a stem cell, a cancer cell or a cell line.

[0015] In one embodiment, the nuclease may be one or more selected from zinc finger nucleases, TALENs (transcription activator-like effector nucleases), and RGENs (RNA-guided engineered nucleases).

[0016] In one embodiment, the nuclease may be Cas9.

[0017] In one embodiment, the polynucleotide encoding the nuclease may comprise one or more selected from a DNA binding domain, a guide RNA, and a cleavage domain.

[0018] In one embodiment, the method can further include a step of introducing a polynucleotide encoding a guide RNA into the cells after the step of introducing a polynucleotide encoding a nuclease into the cells and expressing the nuclease.

[0019] In one embodiment, the exogenous polynucleotide may comprise a polynucleotide encoding a polypeptide or a polynucleotide encoding a functional polyribonucleotide.

[0020] In one embodiment, the functional polyribonucleotide may be one or more selected from the group consisting of microRNA (miRNA), short hairpin RNA (shRNA), Piwi-binding RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and extracellular RNA (exRNA).

[0021] In one embodiment, the exogenous polynucleotide may encode one or more selected from the group consisting of an antibody, an enzyme, a growth factor, a receptor, a hormone, a lymphokine, a cytokine, a signal transduction factor, a reporter, and fragments thereof.

[0022] In one embodiment, the exogenous polynucleotide may be a Sonic hedgehog (SHH) gene.

[0023] In one embodiment, the exogenous polynucleotide may comprise one or more selected from the group consisting of an open reading frame, a polyadenylation sequence, a promoter, an operator, an enhancer, a transcriptional regulator, a signal sequence, and one or more homology regions.

[0024] In one embodiment, after the step of using the nuclease to specifically bind to one or more nucleic acid regions selected from the group consisting of a first nucleic acid region at positions 24894446 to 24894525 on chromosome 9 of the human genome, a second nucleic acid region at positions 9064276 to 9064355 on chromosome 3, and a third nucleic acid region at positions 120174229 to 120174308 on chromosome 4 of the human genome and cleaving the nucleic acid regions, the method may further include the step of introducing into the exogenous polynucleotide a left homology arm (LHA) that binds to a region up to 1 kb to the left of one or more of the cleavage sites of the first to third nucleic acid regions, and a right homology arm (RHA) that binds to a region up to 1 kb to the right.

[0025] In one embodiment, after the step of using the nuclease to specifically bind to one or more nucleic acid regions selected from the group consisting of a first nucleic acid region at positions 24894446 to 24894525 on chromosome 9 of the human genome, a second nucleic acid region at positions 9064276 to 9064355 on chromosome 3, and a third nucleic acid region at positions 120174229 to 120174308 on chromosome 4 of the human genome and cleaving the nucleic acid regions, the method may further include the step of introducing into the exogenous polynucleotide a left homology arm (LHA) that binds to a region up to 0.8 kb on the left side of one or more of the cleavage sites of the first to third nucleic acid regions, and a right homology arm (RHA) that binds to a region up to 0.8 kb on the right side.

[0026] The present invention provides a cell in which an exogenous polynucleotide has been inserted into the genome of the cell by one or more nucleases, and the exogenous polynucleotide may be inserted into one or more of the following nucleic acid regions in the genome of the cell: a first nucleic acid region from bases 24894446 to 24894525 on chromosome 9 of the human genome; a second nucleic acid region from bases 9064276 to 9064355 on chromosome 3; and a third nucleic acid region from bases 120174229 to 120174308 on chromosome 4 of the human genome.

[0027] The present invention provides a composition for expressing an exogenous polynucleotide in a cell, comprising an exogenous polynucleotide and a polynucleotide encoding a nuclease that specifically binds to one or more selected from a first nucleic acid region from positions 24894446 to 24894525 on chromosome 9 of the human genome, a second nucleic acid region from positions 9064276 to 9064355 on chromosome 3, and a third nucleic acid region from positions 120174229 to 120174308 on chromosome 4.

[0028] In one embodiment, the composition may further comprise a left homology arm (LHA) that binds to a region up to 1 kb to the left of one or more of the cleavage sites of the first to third nucleic acid regions, and a right homology arm (RHA) that binds to a region up to 1 kb to the right.

[0029] In one embodiment, the exogenous polynucleotide may be a Sonic hedgehog (SHH) gene.

[0030] The present invention provides a polynucleotide comprising a sequence having at least 90%, 95%, or 99% or more sequence homology to any one of the sequences set forth in SEQ ID NOs: 3 to 5.

[0031] The present invention provides a polynucleotide comprising any one of the sequences set forth in SEQ ID NOs: 3 to 5.

[0032] In one embodiment, the sequence of SEQ ID NO: 3 can specifically bind to a nucleic acid region from positions 24894446 to 24894525 on chromosome 9 of the human genome.

[0033] In one embodiment, the sequence of SEQ ID NO: 4 can specifically bind to a nucleic acid region from positions 9064276 to 9064355 on chromosome 3 of the human genome.

[0034] In one embodiment, the sequence of SEQ ID NO: 5 can specifically bind to a nucleic acid region from positions 120174229 to 120174308 on chromosome 4 of the human genome.

[0035] The present invention provides polynucleotides comprising the sequences of SEQ ID NOs:14 and 15.

[0036] The present invention provides polynucleotides comprising the sequences of SEQ ID NOs:16 and 17.

[0037] The present invention provides polynucleotides comprising the sequences of SEQ ID NOs:18 and 19.

[0038] The present invention provides a vector comprising the polynucleotide.

[0039] [Effects of the Invention]

[0040] The genomic safe harbor of the present invention is characterized by not disrupting the transcription of adjacent genes even when a foreign gene is introduced and by maintaining stable expression of the foreign gene. Therefore, the genomic safe harbor of the present invention can be used for the intracellular expression of various foreign genes.

[0041] [Brief explanation of the drawings]

[0042] [Figure 1] FIG. 1 is a schematic diagram of a gRNA / Cas9 expression vector of the present invention.

[0043] [Figure 2] FIG. 1 is a schematic diagram of a donor vector of the present invention.

[0044] [Figure 3] FIG. 1 is a schematic diagram of the process for constructing gRNA / Cas9 expression vectors targeting AAVS1 and GSH1-3 of the present invention.

[0045] [Figure 4] 1 is a schematic diagram of the donor vector construction process of the present invention.

[0046] [Figure 5] FIG. 1 is a schematic diagram of the process of introducing a foreign gene using the gRNA / Cas9 expression vector of the present invention and a donor vector.

[0047] [Figure 6] 1 is a graph showing the average fluorescence intensity of cells into which the GFP gene was introduced.

[0048] [Figure 7] 1 is a graph showing changes in expression of AAVS and the GSH1 to GSH3 adjacent genes of the present invention.

[0049] [Figure 8] FIG. 1 is a schematic diagram showing the process of introducing the SHH gene into the GSH3 site of ES-MSCs.

[0050] [Figure 9] 1 shows a graph showing SHH mRNA expression in SHH-overexpressing ES-MSCs (SHH-ES-MSCs) and photographs showing hair follicle neogenesis ability.

[0051] DETAILED DESCRIPTION OF THE INVENTION

[0052] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will now be described in detail with reference to the accompanying drawings, so that those skilled in the art can easily understand and practice the present invention. However, the present invention may be embodied in various forms and is not limited to the embodiments and examples described herein.

[0053] Throughout this specification, when a part "comprises" an element, this does not mean that it excludes other elements, but that it may further include other elements, unless otherwise specified.

[0054] The term "genomic safe harbor" as used herein means a region that is capable of maintaining expression of a foreign gene while not disrupting the transcription of adjacent genes.

[0055] The term "nuclease" as used herein refers to a protein used in genome editing or gene editing, including, but not limited to, zinc finger nucleases, TALENs (Transcription Activator-Like Effector Nucleases), and RGENs (RNA-guided engineered nucleases).

[0056] As used herein, the term "exogenous polynucleotide" refers to a polynucleotide that is artificially introduced.

[0057] The term "guide RNA" as used in the present invention means an RNA specific to a target DNA, which binds complementarily to the target DNA and enables a nuclease to cleave the target DNA.

[0058] The term "left homology arm (LHA)" used in the present invention refers to a sequence that can bind to the left end of a site cleaved by a nuclease in human genomic DNA, and is preferably 1000 base pairs, more preferably 800 base pairs.

[0059] The term "right homology arm (RHA)" used in the present invention means a sequence that can bind to the right end of the site cleaved by a nuclease in human genomic DNA, and is preferably 1000 base pairs, more preferably 800 base pairs.

[0060] The term "vector" as used herein may refer to a construct capable of transferring, and preferably expressing, one or more genes or sequences of interest in a host cell, including, but not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as production cells.

[0061] As used herein, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering a composition, and the term "treatment" refers to any action that improves or favorably alters the symptoms of a suspected or affected individual by administering a composition.

[0062] The present invention will be described in more detail with reference to the following examples. However, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0063] [Example]

[0064] [Example 1]

[0065] Construction of vectors for genome safe harbor expression

[0066]

[0067] We prepared a gRNA / Cas9 expression vector (SEQ ID NO: 1) that contains a gRNA sequence, can express the Cas9 protein, and induces DNA double-strand breaks (DSBs) by acting on sequences complementary to the gRNA.

[0068] As shown in Figure 1, the gRNA / Cas9 expression vector contains an origin of replication (ori), a U6 promoter, a gRNA scaffold, a cytomegalovirus promoter (CMV promoter), a chicken beta actin promoter, a Cas9 gene, a puromycin resistance gene, a Poly(A) tail, and an ampicillin resistance gene.

[0069] A donor vector (SEQ ID NO: 2) was prepared to insert a gene sequence encoding a target protein into GSH. In this study, a gene sequence encoding the fluorescent green fluorescent protein (GFP) was inserted and the efficiency of the system was measured. As shown in Figure 2, the donor vector contained a replication origin, a GSH homology region, a puromycin resistance gene, a chicken β-actin promoter, a cytomegalovirus promoter, a GFP gene, a cHS4 insulator, and a kanamycin resistance gene. The homology region was designed to vary depending on the GSH site.

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080] [Table 1] TIFF2025527935000002.tif253170TIFF2025527935000003.tif253170TIFF2025527935000004.tif253170TIFF20255279350 00005.tif253170TIFF2025527935000006.tif253170TIFF2025527935000007.tif253170TIFF2025527935000008.tif217170

[0081]

[0082] [Example 2]

[0083] Selection of Genomic Safe Harbor Candidates

[0084]

[0085] GSH candidates were selected based on the following criteria: cancer-related genes, miRNAs, positions more than 300 kb away from functional small RNAs, positions more than 50 kb away from the 5' end of a gene, positions more than 50 kb away from a replication origin, positions more than 50 kb away from an ultra-conserved region, positions showing low transcriptional activity, positions not contained in regions of copy number variation, positions contained in regions of open chromatin, sequences with only one copy present on human chromosomes, and sequences without TTAA sequences (transposons).

[0086] Next, using the gRNA derivation tools CRISPick, DeepSpCas9, and RGEN, three GSH candidate regions and their corresponding gRNA sequences were identified, as shown in Table 1. CRISPick (Broad Institute) indicates how specifically a gRNA can react to its complementary position on the genome through an on-target score. DeepSpCas9 indicates the efficiency of genome editing by the gRNA through an indel frequency score. RGEN can check for mismatches between the chromosome and the gRNA.

[0087]

[0088] [Table 2]

[0089] [Example 3]

[0090] Construction of a gRNA / Cas9 expression vector targeting genomic safe harbor and AAVS1

[0091]

[0092] gRNA inserts targeting AAVS1, GSH1, GSH2, and GSH3 were introduced into the gRNA / Cas9 expression vector (px459) as follows.

[0093] DNA oligos based on the gRNA insert sequences targeting AAVS1, GSH1, GSH2, and GSH3 were synthesized. The gRNA / Cas9 expression vector was then digested with the restriction enzyme BbsI, and the synthesized DNA oligos were introduced into the linearized gRNA / Cas9 expression vector and ligated. The presence or absence of introduction was confirmed by PCR, and the gRNA sequence introduced into the vector was verified by Sanger sequencing. A schematic diagram of this process is shown in Figure 3.

[0094]

[0095] [Table 3]

[0096] [Example 4]

[0097] Donor vector production

[0098]

[0099] To promote efficient HDR, target-specific homology sequences (≥800 kb) were introduced into the donor vector as follows. Approximately 800 kb above and below the site where DSB occurs by the CRISPR-Cas9 system were designated the left and right homology arms, respectively. Left and right homology arms (SEQ ID NOs: 14-21) corresponding to the genomic locations of AAVS1, GSH1, GSH2, and GSH3 were then synthesized by PCR based on the genome isolated from human adipose stem cells. The donor vector was then digested with the restriction enzymes PmeI and NotI. The homology sequences were introduced into the linearized donor vector using Gibson assembly master mix (NEB). The introduction was confirmed by PCR, and the homologous DNA sequences introduced into the vector were verified by Sanger sequencing. Through the above process, donor vectors of SEQ ID NOs: 22 to 25 were obtained, and a schematic diagram of the above process is shown in FIG.

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

Table 4

[0125]

[0126]

[0127] [Example 5]

[0128] Introduction of GFP expression cassettes from donor vectors into the AAVS1, GSH1, GSH2, and GSH3 positions in the genome and confirmation of expression

[0129]

[0130] 293T cells were used to verify GFP gene transfection and expression, and were cultured in Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% fetal bovine serum (v / v), penicillin (100 U / ml), and streptomycin (100 μg / ml).

[0131] One day before transfection, 5 x 10 cells were transfected into a 24-well culture dish. 4 Dispense 293T cells into aliquots and culture them in a CO2 incubator for 24 hours. After 24 hours, replace with antibiotic-free 293T cell culture medium. Dispense 25 μl of Opti-MEM (Gibco) into each of two 1.5 ml tubes. Mix 650 ng of gRNA / Cas9 vector and 1300 ng of donor vector into one tube (tube 1). Mix 2 μl of Lipofectamine 2000 into the remaining tube (tube 2). Transfer the solution from tube 2 to tube 1, mix, and incubate at room temperature for 10 minutes. After 10 minutes, add approximately 50 μl of the vector-containing solution to 293T cells placed in a 24-well culture dish for transfection. After 48 hours, replace with fresh culture medium.

[0132] 1,000 transfected cells were seeded per 100 mm culture dish, and after two weeks, single-cell-derived colonies expressing GFP were selected. Six, seven, six, and six colonies were selected from 293T cells transfected with GFP expression cassettes into AAVS1, GSH1, GSH2, and GSH3, respectively, and PCR was performed as shown in Figure 5a to confirm the transfer of the foreign gene via HDR. The results are shown in Figures 5b to 5e.

[0133] Furthermore, as shown in Table 5 below, the clones in which gene introduction was confirmed were 16.67%, 42.85%, 50%, and 66.67% for AAVS1, GSH1, GSH2, and GSH3, respectively.

[0134]

[0135] [Table 5]

[0136] To confirm the stability of the transfected GFP gene expression, the mean fluorescence intensity (MFI) of GFP was measured weekly for 4 weeks by flow cytometry. As shown in Figure 6, the MFI values ​​for GSH1, GSH2, and GSH3 transfected with GFP were all higher than those for AAVS1 transfected with GFP.

[0137]

[0138] [Example 6]

[0139] Confirmation of changes in adjacent gene expression

[0140]

[0141] We investigated whether stable expression of a foreign gene could be induced without disrupting the transcription of neighboring genes by introducing the foreign gene into the GSH locus. We analyzed the expression of 10 neighboring genes in AAVS1, 3 in GSH1, 3 in GSH2, and 4 in GSH3 by real-time PCR. The distances between the AAVS1, GSH1, GSH2, and GSH3 loci and the neighboring genes are shown in Table 6.

[0142] Total RNA was extracted from 293T cells transfected with GFP expression cassettes for AAVS1, GSH1, GSH2, and GSH3 using the PureLink RNA Mini Kit (Invitrogen). The extracted RNA was quantified, and complementary cDNA (cDNA) was synthesized using AccuPower RT Master Mix (BIONEER). 100 ng of cDNA was analyzed using TB Green Premix Ex TaqII (Takara) on a QuantStudio 1 Real-Time PCR System (Thermo Fisher). Genes with Ct values ​​above 37 (IZUMO3) were excluded.

[0143] As shown in Figure 7a, the expression of five of the ten genes adjacent to AAVS1 (NLRP2, EPS8L1, TNNI3, SYT5, and PTPRH) was significantly increased or decreased. As shown in Figure 7b, there was no significant difference in the expression of genes adjacent to GSH1. As shown in Figure 7c, there was no significant difference in the expression of NXPH1, a gene adjacent to GSH2. As shown in Figure 7d, there was no significant difference in the expression of any of the four genes adjacent to GSH3.

[0144] Therefore, the GSH1, GSH2, and GSH3 loci not only have higher gene expression efficiency than AAVS1, but also have no effect on the transcription of adjacent genes, which means that they are highly safe when introducing foreign genes.

[0145]

[0146] [Table 6]

[0147]

[0148] [Example 7]

[0149] Evaluation of hair follicle regeneration after introducing the SHH (Sonic hedgehog) gene into the GSH3 site of human embryonic stem cell-derived mesenchymal stem cells (ES-MSCs)

[0150]

[0151] 7.1 Construction of SHH expression cassette

[0152] To introduce the SHH gene into the GSH3 site of ES-MSCs, a donor vector was constructed with the SHH gene sequence inserted instead of GFP, as shown in Figure 8. The donor vector was constructed as follows. First, the GFP gene sequence was removed from the donor vector using a restriction enzyme. Then, an SHH gene fragment was constructed from an SHH expression vector (OriGene) using PCR. The SHH gene fragment was inserted into the donor vector using the Gibson assembly technique. The presence or absence of the fragment in the vector was verified using PCR, and the homologous DNA sequence introduced into the vector was verified using Sanger sequencing.

[0153]

[0154] 7.2 Introduction of an SHH expression cassette into GSH3

[0155] The SHH expression cassette of the donor vector was introduced into the GSH3 site. Experiments were then conducted to confirm the expression of the SHH expression cassette. ES-MSCs were cultured in StemPro MSC SFM XenoFree medium (Gibco) supplemented with L-glutamine (2 mM), penicillin (100 U / ml), and streptomycin (100 μg / ml). The gRNA / Cas9 expression vector and donor vector were combined at a 1:2 ratio, and 2 × 10 cells were electroporated using a Neon electroporation device (Invitrogen). 6ES-MSC cells were transfected with the α-glucanase inhibitor. The overexpression of the SHH gene was verified by real-time PCR. As shown in Figure 9a, SHH mRNA expression in the transfected cells increased approximately 13-fold.

[0156]

[0157] 7.3 Evaluation of the hair follicle neogenesis potential of the SHH expression cassette

[0158] The hair follicle formation potential of SHH-overexpressing ES-MSCs (SHH-ES-MSCs) was evaluated using a patch assay. Epidermal and dermal cells were isolated from the skin of newborn C57BL / 6 mice. ES-MSC spheroids (1 × 10 cells per cell) were cultured in an ultra-low attachment 96-well round bottom plate (S-Bio). 4 100 cells and 1 x 10 epidermal cells 6 The cells were mixed and transplanted subcutaneously into the dorsal skin of 6-week-old nude mice (BALB / cAJcl-nu). The group transplanted with a mixture of epidermal and dermal cells served as a positive control, while the group transplanted with epidermal cells alone served as a negative control. As shown in Figure 9b, hair follicles were formed in the group transplanted with SHH-ES-MSCs 3 weeks after transplantation.

Claims

1. (1) introducing a polynucleotide encoding a nuclease into a cell to express the nuclease; (2) the nuclease specifically binds to and cleaves one or more nucleic acid regions selected from the group consisting of a first nucleic acid region at positions 24894446 to 24894525 on chromosome 9 of the human genome, a second nucleic acid region at positions 9064276 to 9064355 on chromosome 3, and a third nucleic acid region at positions 120174229 to 120174308 on chromosome 4; and (3) introducing an exogenous polynucleotide into the cell and inserting it into the cleavage site of the nucleic acid region; A method for expressing an exogenous polynucleotide in a cell comprising:

2. 2. The method of claim 1, wherein the cells are derived from human blood, body fluids, tissue, stem cells, or carcinomas.

3. 2. The method of claim 1, wherein the cell is a somatic cell, a germ cell, a stem cell, a cancer cell, or a cell line.

4. 2. The method of claim 1, wherein the nuclease is at least one selected from the group consisting of zinc finger nucleases, TALENs (Transcription Activator-Like Effector Nucleases), and RGENs (RNA-guided engineered nucleases).

5. The method for expressing an exogenous polynucleotide in a cell according to claim 4, wherein the nuclease is Cas9.

6. The method for expressing an exogenous polynucleotide in a cell according to claim 1, wherein the polynucleotide encoding the nuclease comprises at least one selected from a DNA binding domain, a guide RNA, and a cleavage domain.

7. 2. The method for expressing an exogenous polynucleotide in a cell according to claim 1, further comprising the step of introducing a polynucleotide encoding a guide RNA into the cell.

8. 2. The method of claim 1, wherein the exogenous polynucleotide comprises a polynucleotide encoding a polypeptide or a polynucleotide encoding a functional polyribonucleotide.

9. 9. The method of claim 8, wherein the functional polyribonucleotide is at least one selected from the group consisting of microRNA (miRNA), short hairpin RNA (shRNA), Piwi-binding RNA (piRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and extracellular RNA (exRNA).

10. The method for expressing an exogenous polynucleotide in a cell described in claim 8, characterized in that the exogenous polynucleotide encodes one or more selected from the group consisting of antibodies, enzymes, growth factors, receptors, hormones, lymphokines, cytokines, signaling factors, reporters, and fragments thereof.

11. The method for expressing an exogenous polynucleotide in a cell according to claim 8, wherein the exogenous polynucleotide is a Sonic hedgehog (SHH) gene.

12. The method for expressing an exogenous polynucleotide in a cell described in claim 8, characterized in that the exogenous polynucleotide comprises one or more selected from the group consisting of an open reading frame, a polyadenylation sequence, a promoter, an operator, an enhancer, a transcriptional regulatory element, a signal sequence, and one or more homology regions.

13. 2. The method for expressing an exogenous polynucleotide in a cell according to claim 1, further comprising the step of introducing into the exogenous polynucleotide a left homology arm (LHA) that binds to a region up to 1 kb to the left of one or more of the cleavage sites of the first to third nucleic acid regions, and a right homology arm (RHA) that binds to a region up to 1 kb to the right of the cleavage site.

14. 14. The method for expressing an exogenous polynucleotide in a cell according to claim 13, further comprising the step of introducing into the exogenous polynucleotide a left homology arm (LHA) that binds to a region up to 0.8 kb to the left of one or more of the cleavage sites of the first to third nucleic acid regions, and a right homology arm (RHA) that binds to a region up to 0.8 kb to the right of the cleavage site.

15. A cell in which an exogenous polynucleotide has been inserted into the genome of the cell by one or more nucleases, A cell characterized in that the exogenous polynucleotide is inserted into one or more of the following nucleic acid regions in the genome of the cell: a first nucleic acid region from bases 24894446 to 24894525 on chromosome 9 of the human genome; a second nucleic acid region from bases 9064276 to 9064355 on chromosome 3; and a third nucleic acid region from bases 120174229 to 120174308 on chromosome 4 of the human genome.

16. A composition for expressing an exogenous polynucleotide in a cell, comprising an exogenous polynucleotide and a polynucleotide encoding a nuclease that specifically binds to one or more selected from a first nucleic acid region from bases 24894446 to 24894525 on chromosome 9 of the human genome, a second nucleic acid region from bases 9064276 to 9064355 on chromosome 3, and a third nucleic acid region from bases 120174229 to 120174308 on chromosome 4.

17. The composition for expressing an exogenous polynucleotide in a cell according to claim 16, further comprising a left homology arm (LHA) that binds to a region up to 1 kb to the left of one or more of the cleavage sites of the first to third nucleic acid regions, and a right homology arm (RHA) that binds to a region up to 1 kb to the right.

18. The composition for expressing an exogenous polynucleotide in a cell according to claim 17, further comprising a left homology arm (LHA) that binds to a region up to 0.8 kb to the left of one or more of the cleavage sites of the first to third nucleic acid regions, and a right homology arm (RHA) that binds to a region up to 0.8 kb to the right of the cleavage site.

19. The composition for expressing an exogenous polynucleotide in a cell according to claim 16, wherein the exogenous polynucleotide is a Sonic hedgehog (SHH) gene.

20. A polynucleotide comprising a sequence having 90% or more sequence homology with any one of the sequences of SEQ ID NOs: 3 to 5.

21. The polynucleotide according to claim 20, comprising a sequence having 95% or more sequence homology with any one of the sequences of SEQ ID NOs: 3 to 5.

22. The polynucleotide according to claim 20, comprising a sequence having 99% or more sequence homology with any one of the sequences of SEQ ID NOs: 3 to 5.

23. The polynucleotide according to claim 20, comprising any one of the sequences of SEQ ID NOs: 3 to 5.

24. The polynucleotide according to claim 20, wherein the sequence of SEQ ID NO: 3 specifically binds to the nucleic acid region from positions 24894446 to 24894525 on chromosome 9 of the human genome.

25. The polynucleotide according to claim 20, wherein the sequence of SEQ ID NO: 4 specifically binds to the nucleic acid region from bases 9064276 to 9064355 on chromosome 3 of the human genome.

26. The polynucleotide according to claim 20, wherein the sequence of SEQ ID NO: 5 specifically binds to the nucleic acid region from bases 120174229 to 120174308 on chromosome 4 of the human genome.

27. A polynucleotide comprising the sequences of SEQ ID NOs: 14 and 15.

28. A polynucleotide comprising the sequences of SEQ ID NOs: 16 and 17.

29. A polynucleotide comprising the sequences of SEQ ID NOs: 18 and 19.

30. A vector comprising the polynucleotide of any one of claims 27 to 29.

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