KR20170030729A - Editing CGG triplet repeats using Endonuclease for Targeting Fragile X mental retardation 1 - Google Patents

Abstract

The present invention relates to the correction of a CGG repeat sequence region using an endonuclease targeted to the FMR1 (Fragile X mental retardation 1) gene and the treatment of Fragile X syndrome using the same. Calibration of the CGG repeat sequence region of the present invention can be accomplished by using an exogenous donor sequence for homologous recombination or using a non-homologous end joining (NHEJ) ) Is a deletion-mediated correction of abnormal CGG repetition.

Description

{Editing CGG triplet repeats using Endonuclease for Targeting Fragile X mental retardation 1} using the endonuclease targeting the FMR1 gene.

The present invention relates to the correction of a CGG repeat sequence region using an endonuclease targeted to the FMR1 (Fragile X mental retardation 1) gene and the treatment of Fragile X syndrome using the same.

Fragile X syndrome (FXS) is the most common genetic disorder of intellectual disability occurring in one out of 3600 men in men and is caused by the silencing of the FMR1 gene located on the X chromosome (1-3 ). The causative mutation of FXS is the expansion of the triple nucleotide CGG repeat (CGG repeat) in the 5'-untranslated region of FMR1 (4-5). Normal persons have CGG repeats of 5 to 55 copies, while patients have more than 200 copies of full mutation (6). The silencing of FMR1 in patients with complete mutations has been demonstrated to be associated with abnormal DNA methylation and epigenetic changes in CGG repeat (7-10).

On the other hand, patient-specific iPSC (induced pluripotent stem cells) derived from diseased persons have been proposed as promising therapeutic resources for the treatment and research of genetic diseases through the correction of causative mutations.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

 Crawford, D. C., Acuna, J. M., and Sherman, S. L. et al. (2001). FMR1 and the fragile X syndrome: human genome epidemiology review. Genet. Med. 3, 359-371  O'Donnell, W.T., and Warren, S.T. (2002). A decade of molecular studies of fragile X syndrome. Ann. Rev. Neurosci. 25, 315-338.  Penagarikano, O., Mulle, J. G., and Warren, S. T. (2007). The pathophysiology of fragile x syndrome. Ann. Rev. Ggenomics Hum. Genet. 8, 109-129  Fuer, Y. H., Kuhl, D. P., Pizzuti, A., Pieretti, M., Sutcliffe, J. S., Richards, S., Verkerk, A. J., Holden, J. J., Fenwick, R. G., Jr., Warren, S. T., et al. (1991). Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox. Cell67,1047-1058  Verkerk, A. J., Pieretti, M., Sutcliffe, J. S., Fu, Y. H., Kuhl, D. P., Pizzuti, A., Reiner, O., Richards, S., Victoria, M. F., Zhang, F. P., et al. (1991). Identification of a gene (FMR-1) containing a CGG repeats coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome. Cell 65,905-914.  Pearson, C. E., Nichol Edamura, K., and Cleary, J. D. (2005). Repeat instability: mechanisms of dynamic mutations. Nat. Rev. Genet. 6, 729-742.  Urbach, A., Bar-Nur, O., Daley, G. Q., and Benvenisty, N. (2010). Differential modeling of fragile X syndrome by human embryonic stem cells and induced pluripotent stem cells. Cell Stem Cell 6,407-411.  A., Yaron, Y., Eden, A., Yanuka, O., Benvenisty, N., R., Urbach, A., Malcov, M., Frumkin, T., Schwartz, T., Amit, A., and Ben-Yosef, D. (2007). Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos. Cell Stem Cell 1,568-577.  Eldar-Geva, T., Schonberger, O., Levy-Lahad, S., Altarescu, G., Renbaum, P., E., Epsztejn-Litman, S., and Eiges, R. (2014). FMR1 epigenetic silencing commonly occurs in undifferentiated fragments of X-affected embryonic stem cells. Stem Cell Rep. 3, 699-706  Tabolacci, E., Moscato, U., Zalfa, F., Bagni, C., Chiurazzi, P., and Neri, G. (2008). Epigenetic analysis reveals an euchromatic configuration in the FMR1 unmethylated full mutations. Eur. J. Hum. Genet. 16, 1487-1498

The present inventors have made efforts to develop an efficient and fundamental treatment method for the fragile X syndrome caused by the CGG repeating base sequence region in the 5'-UTR of the FMR1 gene. As a result, it is possible to successfully remove a CGG trinucleotide having a constant copy number or more by using an RNA-inducing endonuclease that cleaves a guide RNA targeting the upstream region of the CGG repetitive base sequence region and a targeted region And confirming that the removal of such CGG trinucleotide does not affect the ability of stem cells, the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for correcting the CGG repeating base sequence region of the FMR1 gene.

It is another object of the present invention to provide a method for producing induced pluripotent stem cells in which the CGG repetitive base sequence region of the FMR1 gene is corrected.

It is still another object of the present invention to provide a method for producing embryonic stem cells in which the CGG repeating base sequence region of the FMR1 gene is corrected.

Another object of the present invention is to provide a composition for treating Fragile X syndrome.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for correcting a CGG repetitive base sequence region of an FMR1 (fragile X mental retardation 1) gene comprising:

(a) a nucleotide encoding an RNA-derived endonuclease or the RNA-derived endonuclease; And

(b) a nucleotide sequence having a length of 15-25 bp existing in 100 nucleotide sequences located upstream from the starting position of the CGG repeating nucleotide sequence region or a nucleotide sequence of 15-25 bp long nucleotide sequence located downstream from the end position of the CGG repeating nucleotide sequence region (Guiding RNA) that specifically recognizes a nucleotide sequence of 15-25 bp in the nucleotide sequence of 50 or more nucleotides located in the nucleotide sequence of SEQ ID NO:

The present inventors have made efforts to develop an efficient and fundamental treatment method for the fragile X syndrome caused by the CGG repeating base sequence region in the 5'-UTR of the FMR1 gene. As a result, it is possible to successfully remove a CGG trinucleotide having a constant copy number or more by using an RNA-inducing endonuclease that cleaves a guide RNA targeting the upstream region of the CGG repetitive base sequence region and a targeted region , And it was confirmed that the removal of the CGG trinucleotide did not affect the ability of stem cells.

The main experimental results confirmed by the inventors of the present invention are as follows.

We used a RNA-guided endonucleases (RGEN) system to calibrate the CGG repeat site located within the 5'-UTR of FMR1 , and the removal of the CGG repeat was silenced by iPSC (FXS-iPSC) It is possible to induce the re-activation of FMR1 gene expression. We also found that complete DNA demethylation of the FMR1 promoter was induced in the cells of the FXS patient with CGG iterations corrected, and these results indicate that the DNA methylation status of the FMR1 promoter of FXS-iPSC is dependent on the CGG repeat, Of the methylation status of the mice. These studies were the first to confirm that complete mutation of CGG repeat sites in patient-specific iPSC can be corrected using engineered nuclease without the use of orthodontic template DNA. This strategy can be applied to cell therapy for FXS treatment.

As used herein, the term "nucleotide" is a deoxyribonucleotide or ribonucleotide present in single or double stranded form, and includes analogs of natural nucleotides unless otherwise specifically indicated (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

As used herein, the term "specifically recognized" means selectively recognizing it based on complementarity with the target sequence of the present invention and may be used interchangeably with "specifically annealing" have. The term "complementary" means that the inducible RNA of the present invention under conditions of annealing or hybridization is sufficiently complementary to selectively hybridize to the target nucleic acid sequence, and is substantially complementary and completely complementary perfectly complementary).

The term "substantially complementary" means not only a fully matched sequence, but also a sequence that is partially inconsistent between the nucleic acid sequences of the inducible RNA and the FMR1 gene, to the extent that it can be annealed to a particular sequence.

As used herein, the term "CGG repetitive nucleotide sequence region" refers to a site in which the CGG trinucleotide in the 5'-UTR of the FMR1 gene, which is considered to be a pathogen of FXS, appears repeatedly. FXS patients are known to have over 200 copies of the CGG trinucleotide at the CGG repeat sequence. In the present specification, the "CGG repeating base sequence region" has substantially the same meaning as "CGG repeat" or "CGG repeat region", and these terms can be used interchangeably.

As used herein, the term "CGG repetitive nucleotide sequence site" refers to a CGG trinucleotide with a constant number of copies or more by cleavage of the upstream or downstream nucleotide sequence of the CGG repeat nucleotide sequence (Which was present in the region) is removed. Therefore, the term "correction of CGG repeat sequence region" can be regarded as substantially equivalent to "deletion (deletion) of CGG trinucleotide ". Such a deletion of the CGG trinucleotide of a constant copy number, for example, a CGG trinucleotide of 200 copies or more existing in the CGG repeat region of the FXS patient can be corrected to less than 55 copies, which is the range of the normal person.

Also, according to the present invention, demethylation of a CpG island (CpG island) located above the FMR1 gene is induced by removal of the CGG trinucleotide . This demethylation enables the expression of the FMR1 gene.

According to one embodiment of the present invention, the correction of the CGG repeating base sequence region is the removal of a constant copy CGG trinucleotide by cleavage of the upstream nucleotide sequence of the CGG repeat base sequence region.

According to an embodiment of the present invention, the removal of the CGG trinucleotide of the predetermined number of copies results in the presence of 5-55 copies of the CGG trinucleotide at the CGG repeat sequence region of the FMR1 gene.

According to one embodiment of the present invention, the RNA-derived endonuclease is Cas 9 (CRISPR associated protein 9).

As shown in the following examples, CGG trinucleotides above a certain copy number can be removed by cleavage of the nucleotide sequence adjacent to the CGG repeat sequence region in the FMR1 gene. Therefore, the composition of the present invention includes (i) a nucleotide sequence of 15-25 bp in length which is present in 100 nucleotide sequences located upstream from the starting position of the CGG repeating nucleotide sequence region, (ii) A guiding RNA that specifically recognizes a nucleotide sequence of 15-25 bp in the 50 nucleotide sequence located downstream from the end of the nucleotide sequence, or (iii) the nucleotide sequence of the inducible RNA Encoding nucleotides.

According to an embodiment of the present invention, the inducible RNA comprises a nucleotide sequence of 15-25 bp in the nucleotide sequence of the first sequence of Sequence Listing, which is a sequence located upstream from the start position of the CGG repeat sequence region Or inducible RNA that specifically recognizes a complementary sequence thereof.

According to one more specific embodiment, the inducible RNA may be a nucleotide sequence of the second sequence of the sequence listing, which is a sequence located upstream from the start position of the CGG repeat sequence region, or an inducible gene that specifically recognizes the complementary sequence thereof RNA. Sequence Listing The second sequence is the sequence contained in the first sequence of the Sequence Listing.

According to one embodiment, the FMR1 gene to be corrected by the composition of the invention is a FXS FMR1 gene in a patient.

According to another aspect of the present invention, there is provided a method for producing an induced pluripotent stem cell in which a CGG repetitive base sequence region of the FMR1 gene is calibrated, comprising the steps of:

(a) deriving induced somatic cells from patients suffering from fragile X syndrome to obtain induced pluripotent stem cells; And

(b) contacting the inducible pluripotent stem cell with the composition of the present invention or transforming the composition with the gene carrier into which the composition of the present invention is inserted.

According to another aspect of the present invention, there is provided a method for producing embryonic stem cells, comprising contacting embryonic stem cells derived from a patient suffering from fragile X syndrome isolated from an in vitro culture with a composition of the present invention, or transforming a gene carrier into which the composition of the present invention is inserted The present invention provides a method for producing embryonic stem cells in which the CGG repeating base sequence region of the FMR1 gene is corrected.

The induced pluripotent stem cells obtained by reprogramming somatic cells isolated from FXS patients retain the CGG repeat sites in the patient-specific FMR1 gene, which can be calibrated in vitro by the method of the present invention described above. By this calibration, patient-derived guided pluripotent stem cells having a normal range of CGG trinucleotide copy number can be obtained.

As used herein, the term "induced pluripotent stem cell" refers to a cell induced to have pluripotent differentiation potential through artificial reprogramming from differentiated cells, Quot; Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, and specifically have similar cell expression patterns, genes, and protein expression patterns, and are capable of differentiation in in vitro and in vivo , forming a teratoma, Germline transmission is possible. The inducible pluripotent stem cells of the present invention include stem cells derived from all mammals such as human, monkey, pig, horse, cow, sheep, dog, cat, mouse and rabbit. Induced pluripotent stem cells, most specifically inducible pluripotent stem cells derived from FXS patients.

As used herein, the term " de-differentiation "refers to an epigenetic regression process that enables the formation of new differentiated tissues by returning the existing differentiated cells to an undifferentiated state, which is also referred to as a reprogramming process, and the reversibility of epigenetic changes. According to the object of the present invention, the term " de-differentiation "includes all the processes for returning the differentiated cells having a differentiation ability from 0% to 100% to undifferentiated state. For example, The process of differentiating the differentiated cells into differentiated cells having 1% differentiation potential may also be included.

For example, OCT4, NANOG, SOX2, LIN28, KLF4, and c (a) may be added to somatic cells isolated from the FXS patient, and the differentiation may be carried out through various methods known in the art, -MYC. ≪ / RTI >

The medium used for culturing human somatic cells to obtain the induced pluripotent stem cells includes any conventional medium. (Stanner, CP et al., Nat. New Biol. 230: 52 (1971)), Iscove's (Eagle's minimum essential medium, Eagle, H. Science 130: 73: 1 (1950)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio. Med., 73: , CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199: 519 (1967)), F12 (Ham, Proc Natl Acad Sci USA 53: 288 (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8: 396 (1959)), a mixture of DMEM and F12 (Barnes, RG Exp. Cell Res. 29: 515 McCoy ' s 5A (McCoy, TA, et al., ≪ RTI ID = 0.0 > And MCDB series (Ham, RG et al., In Vitro 14: 11 (1978)), and the like, but are not limited thereto no.

As used herein, the term "gene transporter" means a mediator for introducing and expressing a desired target gene into a target cell. Ideal gene carriers are harmless to humans or cells, are easy to mass-produce, and must be capable of delivering genes efficiently.

The term "gene transfer" means that a gene is transferred into a cell and has the same meaning as transduction of a gene into a cell. At the tissue level, the term gene transfer has the same meaning as the spread of a gene. Accordingly, the gene carrier of the present invention can be described as a gene penetration system and a gene diffusion system.

To prepare the gene delivery vehicle of the present invention, the nucleotide sequence of the present invention is preferably present in a suitable expression construct. In such expression constructs, the nucleotide sequence of the invention is preferably operatively linked to a promoter. The term "operably linked" means a functional linkage between a nucleic acid sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, Will control the transcription and / or translation of the other nucleic acid sequences.

The gene carrier of the present invention can be produced in various forms including (i) naked recombinant DNA molecules, (ii) plasmids, (iii) viral vectors, (iv) naturally occurring recombinant DNA molecules or plasmids Or in the form of a liposome or a niozyme.

The nucleotide sequence of the present invention can be applied to all gene delivery systems used for routine animal transformation, including, for example, plasmids, adenoviruses (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 1997), adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retroviruses (Gunzburg WH, et al. Retroviral vectors. Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), lentivirus (Wang G. et al., J. Clin. Invest. 104 (11): R55-62 Viruses (Puhlmann M. et al., Human Gene Therapy 10: 649-657 (1999)), < RTI ID = 0.0 > ), Liposome Human Press 2002) or niosomes.

In the present invention, when the gene carrier is prepared based on a viral vector, the contacting step may be carried out according to a virus infection method known in the art. Infection of host cells with viral vectors is described in the above-mentioned documents.

22: 479 (1980); and Harland and Weintraub, J. Cell Biol. 101: 1094-919), in the case where the gene carrier is a naked recombinant DNA molecule or plasmid in the present invention. 1099 (1985)), calcium phosphate precipitation method (Graham, FL et al., Virology, 52: 456 (1973), and Chen and Okayama, Mol. Cell. Biol. 7: 2745-2752 6: 716-718 (1986)), liposome-mediated transfection (Eugene et al., EMBO J., 1: 841 (1982), and Tur-Kaspa et al., Mol. Cell Biol. 149: 157 (1982), and Nicolau et al., Methods Enzymol., 157: 157 (1982) (1987)) and DEAE-dextran treatment (Gopal, MoI. Cell Biol., 5: 1188-1190 (1985)) and gene bendardment (Yang et al., Proc. Natl. Acad. Sci. 87: 9568-9572 (1990)).

According to still another aspect of the present invention, there is provided an inducible pluripotent stem cell or embryonic stem cell prepared by the above-described method and having a CGG repeating base sequence region of the FMR1 gene corrected.

According to still another aspect of the present invention, there is provided a composition for treating FXS, comprising the inducible pluripotent stem cell or embryonic stem cell of the present invention as an active ingredient.

Since the inducible pluripotent stem cells or embryonic stem cells of the present invention are cells in which the CGG repetitive site of the FXS patient is corrected, when they are differentiated into appropriate somatic cells and transplanted into the patient, they express the corrected gene. Thus, a useful cell therapy composition for FXS, . The compositions of the present invention may be implanted directly into the body, completing the differentiation may be induced to differentiate into the target cell in the transplanted holding vivo, or in vitro to the desired cell back body.

As used herein, the term "treatment" means (a) inhibiting the development of a disease, disease or condition; (b) relief of the disease, disorder or condition; Or (c) eliminating the disease, disease or condition.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers and formulations can be found, for example, in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered parenterally, for example, intravascularly.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . For example, a typical dose of the pharmaceutical composition of the present invention is 10 ² -10 ¹⁰ cells per day on an adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or may be manufactured by intruding into a multi-dose container.

The features and advantages of the present invention are summarized as follows:

(I) The present invention relates to the correction of a CGG repeat sequence region using an endonuclease targeted to the FMR1 gene and the treatment of the fragile X syndrome using the same.

(Ii) Calibration of the CGG repeat sequence region of the present invention can be accomplished by using an exogenous donor sequence for homologous recombination or by using an endonuclease-induced NHEJ (non- homologous end joining (CGCG). This is a deletion-mediated correction.

(Iii) Calibration of the CGG repeating base sequence region using the endonuclease of the present invention does not affect the differentiation ability of stem cells. Therefore, the present invention can be effectively utilized for developing a therapeutic agent for cells using FXS-derived stem cells.

Figures 1A-1F show 5'-UTR sequence analysis and off-target fragmentation analysis of FMR1 . (1a) Schematic drawing of the FMR1 gene 5'-UTR. And the CGG extension site in exon 1. The blue arrow head indicates the PCR primer binding site, and the description of the primers is given in Table 1. (1b) genomic DNA was isolated from each cell. The sequence of CGG extension and its adjacent region was analyzed by Sanger sequencing method. The reference sequence of the FMR1 gene (Ref Sep) was extracted from NCBI (NM_001185082.1). Underlined N indicates an unidentified sequence. (1c) Genomic DNA was isolated from cells transfected with the RGEN-coding plasmid. The PCR product was isolated and sequenced. The RGEN target sequence was indicated in blue and the PAM sequence in red. The red triangle represents the cleavage site, - represents the deleted base, and the lowercase italic chain represents the inserted base. The number of inserted or deleted bases is shown in the right column. (1d) Potential off-target sequences of RGEN used in the experiment were calculated using Cas-OFFinder. Mismatched bases were labeled in blue. The PAM sequence is indicated in red. The names of related genes are shown in the right column. (1e) Four potential off-target sites with high similarity to the RGEN target site were amplified by PCR. To confirm off-target cleavage activity at these positions, T7E1 assays were performed. (1f) allele extension PCR was performed on FXS-iPSC and calibrated FXS-iPSC clones E1 and E3. The analysis showed that FXS-iPSC had more than 200 copies of CGG repeat, whereas no repeat was detected in the corrected FXS clones.
Figures 2a-2f show RGEN induced mutations and reactivation of FMR1 . (2a) The RGEN binding site is shown in the schematic diagram of FMR1 5'-UTR. PAM sequences are shown in red. (2b) Mutations in the RGEN target site were estimated in HEK-293T cells by T7E1 assay. The asterisk indicates the expected location on the DNA band truncated by T7E1. (2c) PCR analysis of genomic DNA of wild-type (WT), patient (FXS) and calibrated (E) cell lines. (2d) The sequence of amplified PCR products from each type of cell was analyzed. Each RGEN target sequence is shown underlined. The PAM sequence is shown in red, the truncated positions are shown in red triangles, the deleted bases are denoted by / and the inserted bases are shown in lowercase italic. FMR1 mRNA expression of wild-type cells (WT), FXS patient cells (FXS) and calibrated clones (El to E3) was analyzed. QPCR was used to detect the expression of FMR1 gene in undifferentiated cell (2e) and expendable NPC (2f). GAPDH expression was used for normalization. The error bar represents the standard error (SE). n is three independent experiments.
Figures 3a-3f show neurovessel formation and FMRP staining of WT and FXS clones. (3a) Bright-field images of nerve roots differentiated from cells by dual Smad suppression. There was no difference in nerve rosette forming ability between the blastocyst and the calibrated clone. Scale bar: 200 占 퐉. (3b) the relative level of expression in APRT, CRYAA and FMR1 FXS and WT iPSC was similar to the expression level of APRT and CRYAA of WT and FXS iPSC, higher level of expression of the FMR1 WT iPSC than the FXS iPSC. (3c) qPCR analysis of pre-multipotency markers shows no significant difference between calibrated clones (E1 or E3) of WT and FXS-iPSC and uncorrected clones. GAPDH expression was used for normalization. The error bar represents the standard error (SE). n = three independent experiments. (3d) Detection of wild-type iPSC (WT), patient ESC (FX), and FMRP in calibrated ESC clones via immunochemistry. Undifferentiated cells grown in a feeder-free medium were fixed and stained with antibody. The DAPI signal (blue) represents the entire cell in the image. Scale bar: 50 탆. (3e) The expression levels of glutamate receptor genes were down-regulated after calibration in FXS-derived neurons. The number on the corrected FXS iPSC rod represents the fraction of the expression level relative to the level of expression of the uncalibrated cells. Expression levels of (3f) core neuronal genes were similar between FXS neurons and calibrated FXS neurons.
Figures 4A-4D show the results of methylation and chromatin conformation analysis of the FMR1 promoter. (4a) Twenty-two CpG sites for use in methylation analysis are shown in the FMR1 promoter from -395 to -256 base pairs from the gene transcription start site. (4b) Pyrosequencing of 22 CpGs of the FMR1 promoter was performed on WT, FXS and calibrated FXS-iPSC (top panel) as well as WT and FXS-NPC (bottom panel) that were not calibrated or calibrated. FXS-iPSC showed complete methylation before gene correction and showed extensive demethylation after correction at most locations (top panel). For NPC, WT and calibrated WT-NPCs showed hypomethylation of the promoter at all analytical sites and FXS-NPCs showed hypermethylation. In contrast to FXS-NPC, the two calibrated FXS-NPC clones showed marked hypomethylation (lower panel), with similar levels of methylation to WT. (4c) Quantification of the methylation level at all CpG sites shows the average methylation rate of each type of cell. WT-iPSC showed hypomethylation and FXS-iPSC showed hypermethylation. After correction of the CGG repeat, FXS-iPSC showed similar low methylation to WT-iPSC (left graph). Similar methylation ratios were confirmed in NPCs differentiated from the aforementioned cells (right graph). (4d) Chromatin immunoprecipitation was performed on FXS-iPSC and calibrated FXS-iPSC. The graph shows relative fold enrichment for chromatin markers. APRT represents open chromatin, CRYAA represents closed chromatin, and FMR1 is compared to APRT and CRYAA . As a result, the FMR1 promoter of FXS-iPSC was similar to the closed chromatin marker, and after the calibration, the promoter was dominated by the open chromatin marker. Error bars indicate SE, * p < 0.05, ** p < 0.005 (by Student's t-test).
Figures 5A-5D show recovery of FMRP in calibrated FXS cells. (5a) Detection of FMRP in wild-type iPSC (WT), patient-derived iPSC (FXS) and calibrated clones (E1 and E3) via immunochemistry. Undifferentiated cells grown in a feeder-free medium were fixed and stained with antibody. The DAPI signal (blue) indicates the total cell presence in the image. Scale bar: 50 탆. (5b) Immunoblot analysis of FMRP in WT, calibrated WT (El), FXS and calibrated FXS clones (El and E3). GAPDH was used as a loading control. (5c) NPC of FMRP of each type of cells was confirmed by immunoblot analysis. GAPDH was used as a loading control. (5d) FMRP detection in wild type (WT), patient (FXS) and calibrated iPSC clones (E1 and E2) via immunochemistry. Mature neurons differentiated for 60 days from each cell line were fixed and stained with antibody. The DAPI signal (blue) indicates the total cell presence in the image. Scale bar: 50 탆.
Figure 6 shows the FMR1 reactivation model. In FXS-iPSC, FMR1 is silenced by the full mutation of the CGG repeat and their methylation, which affects the promoter of FMR1 . After removal of the CGG repeat by RGEN, the promoter is released from its repressive marker and is fully demethylated. Demethylation of the FMR1 promoter allows the gene to be transcribed, thereby FMR1 is reactivated.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Cell culture

HEK-293T cells were grown on Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotic. (Urbach et al., 2010, Cell Stem Cell 6, 407-9258), wild-type iPSC (WT-iPSCs Epi3 line) (Park et al., 2014, Proc. Natl Acad Sci . USA 111, 9253-9258) 411), FXS-derived iPSC (Urbach et al., 2010, Cell Stem Cell 6, 407-411), wild type ESC (Eiges et al., 2007, Cell Stem Cell 1, 568-577), FXS- et al., 2007, Cell Stem Cell 1, 568-577) and calibrated cell lines obtained from the cell lines were maintained in hESC medium containing 4 ng / ml basic fibroblast growth factor (bFGF; PeproTech). To obtain NPCs, nerve rosetts were induced in iPSC and ESC using a dual SMAD-inhibition protocol, as described in the literature (Kim et al., 2010, Stem Cell Rev. 6, 270-281). Were differentiated into neurons as described in the literature (Kim et al., 2012, PLoS ONE 7, e39715). For feeder-free culturing, all cell lines were cultured in Essential 8 ^TM medium (Life Technology).

Cas9 coding plasmids and transfection

A plasmid expressing Cas9 protein and sgRNA was purchased from ToolGen, Inc. (Korea). Potential off-target locations were investigated using Cas-OFFinder (http://www.rgenome.net/; Bae et al., 2014a). Seven potential off-target locations were investigated, of which four were located in exons. To investigate nuclease-induced indels, the off-target positions on the four exons were analyzed with a T7E1 assay. To observe mutagenesis, HEK-293T cells were co-transfected with a plasmid encoding Cas9 and sgRNA using lipofectamine 2000 (Invitrogen). A microporator system (Neon; Invitrogen) was used for the transfection of the plasmid into iPSC or ESC for the correction of CGG repeat. Human iPSC and ESC cultured in the STO cell feeder layer were harvested using collagenase type IV. After washing with PBS, the cells were separated into single cells according to the literature (Desbordes et al., 2008, Cell Stem Cell 2, 602-612). RGEN and sgRNA plasmids were mixed with the single cells and a voltage of 850 V was applied for 30 ms. The cells were redistributed in the feeder layer and allowed to grow for 10 days. To detect CGG repeats genomic editing, the cells were lysed and PCR was performed as in the literature (Park et al., 2014, Proc. Natl Acad Sci USA 111, 9253-9258) . The primer sequences used are shown in Table 1.

T7E1 assay

Four days after co-transfection with Cas9 and sgRNA plasmids, genomic DNA was isolated and purified from transfected cells using a DNeasy Tissue kit (Qiagen). Then, the DNA fragment containing the nuclease target site was amplified using the primer set of Table 1. (Guschin et al., 2010, Methods Mol. Biol. 649, 247-256). The amplified duplex DNA was denatured and annealed to generate heteroduplex DNA fragments. Thereafter, the sample was treated with T7 endonuclease 1 at 37 占 폚 for 20 minutes to break the mismatch position and analyzed by agarose gel electrophoresis to determine the band intensity of the uncut (uncorrected) section and the cut section Respectively. The mutation rate was calculated using the equation, "(1-parental fraction) x 100 ".

Isolation and PCR analysis of clonal cells

To isolate clonal populations of calibrated cells, each colony identified by PCR as having deletion in CGG repeat was isolated into single cells and then redistributed into a new feeder layer. After three passages, two to three clones were selected for sequencing and further experiments. In order to confirm the sequence of the CGG repetition, genomic DNA including the upstream region and the downstream region was subjected to PCR analysis using Ex-Taq polymerase (Takara) and the primers shown in Table 1. LA-taq and GC buffer I (Takara) were used to determine the full sequence of CGG repeats in HEK-293T, WT-iPSC and WT-ESC. The amplified PCR product was electrophoresed and then eluted with a baby goethite gel. Sequences of the eluted DNA fragments were analyzed using the primers used in PCR amplification. Analysis of CGG repeat lengths of FXS iPSC and calibrated FXS iPSC clones E1 and E3 was performed using the AmplideX FMR1 PCR kit (Asuragen).

RNA isolation, RT-PCR and microarray

Total RNA was isolated from the cells using TRIzol reagent (Invitrogen). 1 ㎍ of total RNA was used for cDNA synthesis using DiaStar ^TM cDNA synthesis kit (SolGent, Korea). To confirm FMR1 expression, PCR was performed with EmeraldAmp PCR Master Mix (Takara) using the synthesized cDNA as a template. In order to quantify the level of FMR1 mRNA, using SYBR ^ⓡ PremixEx-Taq (Takara) it was subjected to qPCR. For FMR1 mRNA amplification, a forward primer ( FMR1- qF) located in exon 3 and a reverse primer ( FMR1- qR) located in exon 4 were used (Chiurazzi et al., 1999, Hum. Mol. Genet. , 2317-2323). The sequences of the primers used in qPCR are shown in Table 1 above.

For microarray analysis, RNA was extracted according to the protocol of the manufacturer (Affymetrix). RNA was used for Human Gene 1.0 ST microarray platform (Affymetrix) analysis, and washing and scanning were performed according to the manufacturer's protocol. The arrays were analyzed using the Robust Multichip Analysis (RMA) of the Affymetrix Expression Console.

DNA methylation analysis

For DNA methylation analysis, genomic DNA was isolated and purified using the NucleoSpin Tissue kit (Macherey-Nagel). Pyrosequencing was performed with an EpigenDx (Hopkinton, Mass.) Using a primer set targeting the 22 CpG positions of the FMR1 promoter.

ChIP (Chromatin immunoprecipitation)

For ChIP, FXS and calibrated FXS-iPSC were pooled, fixed, crosslinked with formaldehyde solution, dissolved and sonicated. Salmon sperm agarose beads (Millipore) were treated at 4 ° C for 1 hour to clear chromatin. Immunoprecipitation of chromatin was performed with anti-acetylated histone H3 antibody (Millipore 06599), anti-methylated histone H3 antibody (Millipore 17614) and anti-methylated histone H3 (Millipore 17648) &Lt; / RTI &gt; overnight. After overnight incubation, cross-linking was reversed and DNA was recovered using PCR clean-up kit (Qiagen). The eluted DNA fragments were used for quantitative PCR analysis. Primers for the FMR1 promoter, the APRT promoter (positive control) and the CRYAA promoter (negative control) were used for the PCR analysis. The primer sequences used in ChIP qPCR are shown in Table 1.

Western blot analysis

Cells were lysed in ice for 20 minutes using RIPA buffer containing protease inhibitor mixture (Roche). The protein concentration was measured by Bradford assay (Bio-Rad). Then, the same amount of protein was separated by electrophoresis and transferred to PVDF (polyvinylidene fluoride) membrane (Millipore). After blocking with 5% non-fat skimmed milk dissolved in PBS, the membranes were incubated with Tris-buffered saline (20 mM) containing 0.05% Tween 20 with anti-FMRP antibody (AbFrontier, Korea) and anti- GAPDH antibody Tris-HCl and 500 mM NaCl) at room temperature for 2 hours. After two washes in Tris-buffered saline / Tween, the membranes were incubated in Tris-buffered saline / Tween with horseradish peroxidase-conjugated goat anti-mouse antibody for 1 hour at room temperature. Proteins were detected using an enhanced chemiluminescence kit (Amersham Biosciences).

Immunodiffusion

For immunostaining of FMRP, cells were fixed with 4% paraformaldehyde solution and then permeabilized with 0.2% Triton X-100. After blocking with PBS solution containing 5% normal goat serum and 2% bovine serum albumin, cells were incubated with anti-OCT3 / 4 (Santa Cruz), anti-FMRP (AbFrontier, Korea) and anti-MAP2 Lt; RTI ID = 0.0 &gt; 1 &lt; / RTI &gt; After washing three times with PBS solution, cells were incubated with fluorescence-conjugated secondary antibody (Alexa Fluor 488 or 594; Invitrogen) for 1 hour at room temperature. Then, the cells were washed with a PBS solution containing 0.1% Tween 20 and placed on a mounting medium containing 4 ', 6-Diamidino-2-phenylindole (DAPI; Vector Laboratories) for observation of nuclei. Images were captured and analyzed using an Olympus IX71 microscope or FSX system.

Statistical analysis

Data are expressed as mean ± standard error (SE). The significance of the data was assessed by Student's t test. p <0.05 was considered statistically significant.

Experiment result

5'-UTR sequence analysis of FMR1 gene

Sequences of CGG repeats of wild-type (WT) - and FXS-iPSC and ESC and their adjacent regions (upstream and downstream of CGG repeat) were analyzed to find DNA sequences in the FMR1 gene that would enable the deletion of CGG repeats . First, the 5'-UTR was amplified from WT cell lines using the FMR1-F and FMR1-R primer sets (FIG. 1A). In the case of FXS-iPSC and FXS-ESC, the upstream and downstream regions of the CGG repeat were individually amplified using FMR1-F / FMR1-R1 and FMR1-F1 / FMR1-R, respectively (FIG. The reason for this is that it is difficult to amplify over 200 copies of completely mutated CGG repeats found in FXS patients by conventional PCR methods. In fact, FXS-iPSC used in this experiment has more than 450 CGG repeats, and FXS-ESC used in this experiment has more than 200 CGG repeats (Gray et al., 2007, Mol. Cell. Biol., 27, 426-437; Eiges et al., 2007, Cell Stem Cell 1, 568-577). Our WT cells had a repeating structure of 9-10 CGG trinucleotides with or without one AGG trinucleotide (Figure 1b, Table 2). Interestingly, the sequences of adjacent upstream and downstream regions in WT and patient derived cells were identical (FIG. 1 b).

cell The number of copies of the CGG iteration Ref (NCBI # NM_001185082.1) CGG (10) AGG (1) CGG (9) HEK-293T CGG (10) AGG (1) CGG (9) AGG (1) CGG (9) WT-iPSC CGG (9) AGG (1) CGG (19) WT-iPSC1 CGG (9) AGG (1) CGG (10) AGG (1) CGG (9) WT-ESC CGG (10) AGG (1) CGG (9) AGG (1) CGG (9) FXS-iPSC Over 200 copies * FXS-ESC Over 200 copies **

Numbers in parentheses indicate the number of repeated sequences.

The number of copies of FXS-iPSC is described in the literature Gray, SJ, et al . An origin of DNA replication in the promoter region of the human fragile X mental retardation (FMR1) gene. Mol. Cell. Biol. 27, 426-437 (2007).

** The number of copies of FXS-ESC is described by Eiges, R., et al. Developmental study of fragile X syndrome using human embryonic stem cells derived from preimplantation genetically diagnosed embryos. Cell Stem Cell 1, 568-577 (2007).

RGEN-induced mutations in the FMR1 gene in HEK-293T cells

Next, RGEN consisting of sgRNA targeting the Cas9 nuclease and the end of the upstream sequence of the CGG repeat was prepared (Fig. 2a). To verify the genome-editing activity of RGEN, T7 endonuclease 1 (T7E1) assays were performed on HEK-293T cells. RGEN activity was relatively high, leading to a mutation at a frequency of 41% at the target site (Fig. 2b). In subsequent sequencing, large deletion sequences as well as small insertions and indels were identified at the RGEN target site (FIG. 2C). Considering that off-target mutations can have undesirable effects when using the RGEN system (Fu et al., 2013, Nat. Biotechnol. 31, 822-826; Hsu et al., 2013, Nat. 201, Genome Res. 24, 132-141), and these mutant types were used in the RGEN system used in this experiment To determine whether or not it was caused by. Four potential off-target positions similar to the on-target positions in the human genome were examined using the Cas-OFFinder program (Fig. 1d). Thereafter, it was verified by T7E1 analysis that the RGEN system used in this experiment did not produce any detectable off-target mutations at these positions (Fig. 1e).

Targeted genomic calibration of CGG repeat in patient's iPSC and ESC

To correct FXS in patient cells, the designed RGEN was used to calibrate FXS-iPSC and FXS-ESC. Cas9 and sgRNA plasmids were electroporated with WT cells, FXS-iPSC and FXS-ESC, and PCR-based genotyping was performed to screen the calibrated clones. Of all calibrated WT-iPSC and WT-ESC, clones with CGP repeat deletions of approximately 90 bp compared to the parental lines were selected for use as controls in subsequent experiments (Table 2). In FXS-iPSC and FXS-ESC, clones with PCR products of similar size to the PCR products of only 90 bp deleted WT cells were selected (Fig. 2C). After screening and additional passaging, 2-3 calibrated cells from each cell type with CGG repetitive deletion were constructed (~ 2-3% efficiency) in about 100 colonies and confirmed by PCR-based genetic characterization 2c). All calibrated cells showed deletion of the CGG repeat sequence in the 5'-UTR of FMR1 (FIG. 2d). These results demonstrate that targeted DSB (DNA double-strand break) upstream of the CGG repeat was induced by Cas9 nuclease and could significantly delete the abnormally mutated repeat. Finally, the corrected FXS-iPSC clones E1 and E3 Our FXS-iPSC as well as to verify the complete resection of the CGG repeat in the selected corrected FXS-iPSC (ablation) is also using the AmplideX ^ⓡ FMR1 PCR Kit Respectively. Analysis showed that the parental FXS-iPSC had more than 200 CGG repeats, whereas no CGG was detected in the corrected FXS iPSC clones (Fig. 1F).

Resumption of FMR1 gene expression in calibrated FXS-iPSC and FXS-neuronal precursor cells (NPC)

Based on the genomic DNA calibration results, it was analyzed whether deletion of CGG repeat resulted in reactivation of FMR1 . To assess the reactivation of the silenced FMR1 gene, mRNA levels were measured in qPCR assays in calibrated or uncorrected isogenic cell lines in WT- and FXS-iPSC. Unlike the iPSC model of patients with FXS, the expression of FMR1 remained active in FXS-ESC, and silencing was occasionally obtained only after very long differentiation into mature neurons (Urbach et al., 2010, Cell Stem Cell 6, 407-411). Because of these differences between patient-derived iPSCs and ESCs, we focused on the reactivation of the FMR1 gene by genomic correction in the iPSC model system. Interestingly, in the calibrated FXS-iPSC (FXS-iPSC clones E1 and E3), the reactivation of the silenced FMR1 mRNA occurred after the deletion of the CGG repeat. In addition, the level of FMR1 mRNA was restored similar to that observed in control WT cells. Furthermore, the deletion did not show any major change in the transcription of FMR1 in WT-iPSCs calibrated compared to the uncalibrated parental strain (Fig. 2E).

Nerve roots were induced from WT, FXS-calibrated and FXS-uncorrected homozygous cell lines to determine if the reactivation identified in iPSC persisted through neural differentiation. There was no difference in the rate of neural rosette formation between the calibrated iPSC and ESC when compared to their parental cells, and these results show that all cell lines used in this experiment have similar potential to differentiate into early neuronal cells 3a). Transcription of the FMR1 gene was maintained in NPCs differentiated from all calibrated FXS-iPSC cell lines (mainly FXS-iPSC E1, E2 and E3), similar to the expression level of WT-iPSC (Fig. 2f).

Analysis of methylation status of promoter region of FMR1

After confirming the successful reactivation of the RNA levels of FMR1, it was to determine whether the change in the mRNA expression is related to the welfare enemies (epigenetic) changes in the promoter region of the FMR1. First, the methylation status of the FMR1 promoter of iPSC was analyzed to determine whether the deletion of CGG repetition affects the CpG island (CpG island) located upstream of the promoter. Using pyrosequencing, 22 CpG sites between -395 and -256 bp from the start of FMR1 transcription were analyzed (FIG. 4A). As a result, all CpG sites were hyper-methylated (Fig. 4B), as most CpG sites showed 100% methylation before deletion of CGG repeat in FXS-iPSC by RGEN. Analysis of the 22 CpG sites at the same positions of the FMR1 promoter in the calibrated FXS-iPSC clones revealed that the methylation level of many CpG sites was similar to the methylation level found in WT cells, indicating a significant demethylation of the promoter (Fig. 4B). As a result of quantifying the result of pirosequencing, the average methylation level of the WT-iPSC promoter was 0.4%, and the average promoter methylation of FXS-iPSC reached 97.4%. Analysis of the calibrated FXS-iPSC revealed that the FXS-iPSC clone E1 was 10% and the FXS-iPSC clone E3 was only 0.8%, similar to WT-iPSC, indicating that the mean methylation of the promoter was significantly reduced (Fig. 4c).

The levels of FMR1 promoter methylation of NPCs from the WT, FXS, calibrated WT and calibrated FXS-iPSC were analyzed. Similar to that observed in iPCS, NPCs also showed marked changes in the level of promoter methylation between the calibrated FXS clones and the uncalibrated FXS clones. Although WT-NPC corrected WT-NPC showed low methylation with almost no methylation at all sites, FXS-NPC showed mostly 100% methylation (hypermethylation) at most sites (FIG. 4b). In the calibrated clones, both FXS-NPC clones were markedly under-methylated with almost no methylation at their positions (FIG. 4B). As a result of quantifying all sites, the mean methylation of the WT-corrected WT-NPC FMR1 promoter was 0.2% and 0.7%, respectively (FIG. 4c). The average promoter methylation level of FXS-NPC was 87.4%, and the average promoter methylation level of the corrected FXS-NPC was only 0.2% in both clones, similar to that of WT-NPC (FIG.

To analyze the changes in the chromatin structure of the FMR1 gene, histone 3 tail acetylation (H3 Ace) and histone 3 K4 methylation (histone 3 K4 methylation, which shows the transcriptionally active chromatin status) H3 K4meth), and chromatin immunoprecipitation against histone 3 K9 methylation (H3 K9 methylation; H3 K9meth) associated with FMR1 silencing, which showed repressed chromatin status Coffee et al., 2002, Am. J. Hum Genet., 71, 923-932, Coffee et al., 1999, Nat. Genet., 22, 98-101). The two markers for activated chromatin, H3 Ace and H3 K4meth, were significantly up-regulated after the correction of the CGG repeat and the inhibited chromatin marker H3 K9meth was significantly down-regulated (Figure 4d). The expression levels of APRT , CRYASA and FMR1 in WT and FXS-iPSC were also analyzed by RT-PCR. The expression analysis confirmed the correlation between expression and epigenetic modification (Fig. 3B). These results show that the elimination of CGG repeats has a widespread genetic influence on the methylation status and chromatin status of the FMR1 promoter.

Recovery of FMRP Levels in Corrected FXS Clones

Finally, we investigated the presence of FMR1 protein (FMRP) through immunocytochemical staining of calibrated iPSC cell lines and their parental cells. As expected, staining of undifferentiated FXS-iPSC was negative for FMRP as opposed to that WT-iPSC staining was positive for FMRP (Fig. 5A). The above results were further proved by Western blotting, which allows more sensitive detection of FMRP (Fig. 5B). Similar to the transcriptional and epigenetic results described above, FMRP levels were restored in the calibrated FXS-iPSC E1 and E3. All calibrated cell lines showed OCT4 staining similar to their parental cells (Fig. 5A). Further, other pluripotency markers showed similar expression levels in both calf cells and their progenitor cell lines as a result of qPCR analysis (Fig. 3c). These results show that the procedure that results in the deletion of CGG repeats does not adversely affect the ability of the calibrated clones to differentiate. Further analysis of the calibrated ESC cell lines showed FMRP staining similar to their isogenic ESCs (Fig. 4d). Next, the presence of FMRP was analyzed by Western blotting on NPCs derived from FXS-iPSC clones E1 and E3, and a high level of FMRP was identified (FIG. 5C).

We investigated whether the presence of FMRP was also maintained in mature neurons derived from calibrated FXS-iPSC clones. To this end, we have differentiated the WT- and FXS-iPSC as well as the calibrated cell line of the homozygous genotype into mature neurons (Kim et al., 2010, Stem Cell Rev. 6, 270-281; Kim et al., 2012 , PLoS ONE 7, e39715). The differentiated neurons were then stained for FMRP and for mature neuron marker MAP2 (microtubule-associated protein 2). All differentiated cell lines were stained positively for MAP2 and these results show successful differentiation into mature neurons (Fig. 5d). Fortunately, mature neurons differentiated from FXS-iPSC clones E1 and E2 were positively stained for FMRP (Fig. 5d), unlike neurons differentiated from unmasked FXS-iPSC. To investigate the potential effects of reactivated proteins (FMRPs), gene expression of calibrated FXS mature neurons was compared to neurons from parental strains of their homozygous genotypes. The gene expression analysis identified some genes differentially expressed between the calibrated FXS neurons and the uncalibrated FXS neurons, and these genes contained many different glutamate receptor genes. Some glutamate receptor genes showed a decrease in RNA levels by more than 2-fold after calibration (FIG. 3E), while other core neuron genes showed similar expression levels (FIG. 3F). These results are related to previous studies (Dolen et al., 2007, Neuron, 56, 955-962) showing dysregulation of glutamate receptor activity in FXS neurons.

Collectively, the above results show that genetic modification through deletion of abnormal repetitions can restore the expression of the FMR1 gene and the FMRP levels in FXS-iPSC and their neural derivatives.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION, Yonsei University <120> Editing CGG triplet repeats using Endonuclease for Targeting          Fragile X mental retardation 1 <130> PN150352 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 87 <212> DNA <213> Artificial Sequence <220> <223> potential RGEN target site in upstream region of CGG repeat <400> 1 cttccggtgg agggccgcct ctgagcgggc ggcgggccga cggcgagcgc gggcggcggc 60 ggtgacggag gcgccgctgc caggggg 87 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RGEN target site in upstream region of CGG repeat <400> 2 tgacggaggc gccgctgcca 20 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> FMR1-F <400> 3 tcaggcgctc agctccgttt cggtttca 28 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> FMR1-R <400> 4 aagcgccatt ggagccccgc acttcc 26 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMR1-F1 <400> 5 cggcggcggc tgggcctcga g 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMR1-R1 <400> 6 ccgccgccgc gctgccgcac g 21 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CDC42BPG-F <400> 7 cctgcgccat ccacgtagat gccagca 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> CDC42BPG-R <400> 8 gcagacatct tccaggtggg ggagtgc 27 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> VPS39-F <400> 9 cagggcacaa gatagccagt aatgtcc 27 <210> 10 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> VPS39-R <400> 10 agaattagcc tgaaacctgt tctgatg 27 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> ELAVL1-F <400> 11 caggtgtccc tgcaccccct ttgtgac 27 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> ELAVL1-R <400> 12 gtttccacat cctggaggag gggt 24 <210> 13 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GCNT7-F <400> 13 caggagaatc gcttgaaatc aggaggc 27 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> GCNT7-R <400> 14 caagacgctg acccatgaat gccacga 27 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F <400> 15 gaacatcatc cctgcctcta ctg 23 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R <400> 16 caggaaatga gcttgacaaa gtgg 24 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F1 <400> 17 cccctcaagg gcatcctggg cta 23 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R1 <400> 18 gaggtccacc accctgttgc tgta 24 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> FMR1-qF <400> 19 gtatggtacc atttgttttt gtg 23 <210> 20 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> FMR1-qR <400> 20 catcatcagt cacatagctt ttttc 25 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FMR1 promoter F <400> 21 aactgggata accggatcga t 21 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> FMR1 promoter R <400> 22 ggccagaacg cccatttc 18 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> APRT promoter F <400> 23 gccttgactc gcacttttgt 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> APRT promoter R <400> 24 taggcgccat cgattttaag 20 <210> 25 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CRYAA promoter F <400> 25 ccgtggtacc aaagctga 18 <210> 26 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CRYAA promoter R <400> 26 agccggctgg ggtagaag 18

Claims (11)

Composition for correcting CGG repeating base sequence region of FMR1 (fragile X mental retardation 1) gene comprising:
(a) a nucleotide encoding an RNA-derived endonuclease or the RNA-derived endonuclease; And
(b) a nucleotide sequence having a length of 15-25 bp existing in 100 nucleotide sequences located upstream from the starting position of the CGG repeating nucleotide sequence region or a nucleotide sequence of 15-25 bp long nucleotide sequence located downstream from the end position of the CGG repeating nucleotide sequence region (Guiding RNA) that specifically recognizes a nucleotide sequence of 15-25 bp in the nucleotide sequence of 50 or more nucleotides located in the nucleotide sequence of SEQ ID NO:
The composition of claim 1, wherein the RNA-derived endonuclease is Cas 9 (CRISPR associated protein 9).
3. The method according to claim 1, wherein the inducible RNA comprises a nucleotide sequence of 15-25 bp in length, or a complement thereof, present in the nucleotide sequence of the first sequence of Sequence Listing which is a sequence located upstream from the start position of the CGG repeat sequence region Lt; RTI ID = 0.0 &gt; a &lt; / RTI &gt; sequence.
[Claim 2] The method according to claim 1, wherein the inducible RNA is a nucleotide sequence of the second sequence of SEQ ID NO: 2, which is a sequence located upstream from the start position of the CGG repeat sequence region or an inducible RNA that specifically recognizes a complementary sequence thereof &Lt; / RTI &gt;
The composition of claim 1, wherein the FMR1 gene is an FMR1 gene in a patient suffering from Fragile X syndrome.
2. The composition of claim 1, wherein the calibration of the CGG repeat sequence region is the removal of a constant copy CGG trinucleotide by cleavage of the upstream nucleotide sequence of the CGG repeat sequence region.
7. The composition according to claim 6, wherein a CGG trinucleotide of 5-55 copies is present in the CGG repetitive base sequence region of the FMR1 gene by removal of the CGG trinucleotide of the constant copy number.
7. The composition of claim 6, wherein demethylation is induced in a CpG island located upstream of the FMR1 gene by removal of the CGG trinucleotide .
A method for producing induced pluripotent stem cells in which a CGG repetitive base sequence region of FMR1 (fragile X mental retardation 1) gene including the following steps is corrected:
(a) deriving induced somatic cells from patients suffering from fragile X syndrome to obtain induced pluripotent stem cells; And
(b) contacting the inducible pluripotent stem cell with the composition of any one of claims 1 to 8, or transforming with the gene carrier into which the composition is inserted.
Comprising contacting a cell with a composition of any one of claims 1 to 8 with an embryonic stem cell derived from an ex vivo separated fragile X syndrome patient or transforming with the gene carrier into which said composition is inserted, fragments X mental retardation 1) A method for producing embryonic stem cells in which the nucleotide sequence of CGG repeats is corrected.
A composition for treating fragile X syndrome comprising the induced pluripotent stem cells prepared by the method of claim 9 or the embryonic stem cells prepared by the method of claim 10 as active ingredients.

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