KR101658135B1 - Endonuclease for Targeting blood coagulation factor and Use Thereof - Google Patents

Abstract

The present invention relates to a method for inversion of normal blood coagulation factor VIII (F8) gene, a method for inversion of blood coagulation factor VIII (F8) gene inversion, and a method for inversion Stem cells.
The method of the present invention is a research tool for studying the pathogenesis of type A hemophilia and for screening therapeutic drugs by effectively reproducing the inverse of intron 1 and intron 22 of F8 gene which accounts for a large number of causes of severe A hemophilia Can be usefully used. In addition, the inverse-proof inducible pluripotent stem cells prepared by the method of the present invention are effective for restoring type-A hemophilia by restoring the mutant genotype to a wild-type state without restricting the normal gene or protein transfer It enables the fundamental treatment.

Description

BACKGROUND OF THE INVENTION Endo-nucleases targeting the blood coagulation factor VIII gene and their uses {Endonuclease for Targeting Blood Coagulation Factor VIII and Use Thereof}

The present invention relates to a composition for treating type A hemophilia using an endocervical agent targeting a blood coagulation factor VIII gene.

Hemophilia A is one of the most common genetic bleeding disorders worldwide with a prevalence of 1 in 5,000 men. This disease is caused by various gene mutations in blood coagulation factor VIII (F8). Depending on the genotype, clinical symptoms are divided into mild (5-30% active), moderate (1-5% active) and severe (<1% active) (Graw et al., 2005). Approximately half of severe hemophilia A includes intron homologue F8 1 (int1h, about 5% of severe A-type hemophilia) and 22-intron homolog (int22h, about 40% of severe A-type hemophilia) rather than point mutation It is caused by chromosome inversion. These two inversions result from incorrect restoration of homologous DNA double-strand breaks (DSBs) through nonallelic homologous recombination (NAHR). Previously, the present inventors prepared customized wild-type human cell lines in order to invert the chromosomal segments between two identical int1h sequences (int1h-1 and int1h-2) that are 140kp apart and have a frequency of 0.1% and 1.9% One zinc finger nuclease (ZFN) has been used in transcriptional activator-like effect nuclease (TALEN) in induced pluripotent stem cells (iPSC) (Park et al., 2014). This method artificially induces inverted genotypes mimicking an erroneous DSB restoration. Other 600-kbp inverted sequences containing three int22h sequences (int22h-1, -2 and -3) were 8 times more frequent than the 140-kbp inversion, but the size of the inverted region was large and not two on the X chromosome There existed homologues, which made recovery more difficult. In addition, int22h (10 kbp) is much larger than int1h (1 kbp), so it is very difficult to analyze the genotype of the int22h inversion or repair using conventional PCR, since the entire 10-kbp int22h must be amplified . In fact, it is not known that a 600-kbp chromosome segment containing int22h in human cells, as well as patient-derived iPSCs, can be restored using programmable nuclease.

We have shown that RNA-guided engineered nuclease (RGEN) (Cho et al., 2013; Cho et al., 2014) as a short regressive repetitive Cas (CRISPR) ) To restore normalization of these two inversions in iPSCs derived from patients with type A haemophilia.

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.

The present inventors have made extensive efforts to develop an effective and fundamental treatment method for hemophilia type A induced by various gene mutations in blood coagulation factor VIII (F8). As a result, it has been found that the inversion of the F8 gene can be successfully corrected using a guide RNA targeting the inversion site and an RNA-induced endonuclease that cleaves the site targeted thereby, .

Accordingly, an object of the present invention is to provide a composition for inversion of blood coagulation factor VIII (F8) gene and an inversion correction method using the same.

Another object of the present invention is to provide a method for producing inducible pluripotent stem cells in which the inverse of blood coagulation factor VIII (F8) gene is calibrated, inducible pluripotent stem cells prepared therefrom, and A And to provide a composition for treating hemophilia.

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 inversion of blood coagulation factor VIII (F8) gene comprising:

(a) RNA-derived endonuclease or RNA-derived endonuclease encoding nucleotides; And

(b) a guiding RNA that specifically recognizes the following cleotide sequence or a complementary sequence thereof, or a nucleotide encoding the derived RNA:

(I) a nucleotide sequence of SEQ ID NO: 1;

(Ii) the nucleotide sequence of SEQ ID NO: 2; And

(Iii) a 15-25 bp nucleotide sequence present in nucleotides 562,975 bp in length between the above (i) and the above (ii) on the 22nd intron of the F8 gene

(Iii) is at least 80% identical to (i) or (ii) in the 562,975 bp long nucleotide between (i) and (ii) Wherein no nucleotide sequence with sequence homology is present and 5 &apos; -N (G / A) G-3 &apos; nucleotides are linked downstream.

The present inventors have made extensive efforts to develop an effective and fundamental treatment method for hemophilia type A induced by various gene mutations in blood coagulation factor VIII (F8). As a result, it has been found that the inversion of the F8 gene can be successfully corrected using an RNA-induced endonuclease that cleaves a target RNA and a targeting RNA targeting the site of inversion.

According to the present invention, a target site is guided using guiding RNA that specifically recognizes the inversion site of intron 22 of intron 22 of F8 gene, and the site is referred to as RNA- By inverting the inducible endonuclease, the inverted gene region is cleaved by reversal. The cleaved gene site causes re-circulation at a certain frequency, resulting in correction of the inversion.

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

The term &quot; specifically recognized &quot; in the present invention means selectively recognizing it based on complementarity with the target sequence of the present invention, and is used interchangeably with &quot; specifically annealing &quot;. The term &quot; complementary &quot; means within the scope of the present invention that the inducible RNA of the present invention selectively hybridizes to the target nucleic acid sequence under the desired annealing or hybridization conditions, substantially complementary and fully perfectly complementary &quot; and &quot; complementary &quot;, and specifically, the term &quot; complementary &quot; As used herein, the term &quot; substantially of the present invention &quot; means not only a completely matched sequence but also a sequence partially inconsistent with the sequence to be compared within a range that can be annealed to a specific sequence.

According to the present invention, the first sequence of the sequence listing of the present invention is characterized in that there is no other nucleotide sequence having 80% or more sequence similarity among 20 bp consecutive nucleotide sequences present downstream of int22h-1 of the F8 gene, (G / A) G-3 'nucleotide directly to the 5'-N (G / A) G-3' nucleotide sequence, and the second sequence of the sequence listing comprises a 20bp contiguous nucleotide sequence present upstream of int22h-3 of the F8 gene (G / A) G-3 'nucleotide directly upstream of the 5' -N (G / A) G-3 'nucleotide sequence without any other nucleotide sequence having 80% or more of sequence similarity. Therefore, when a nucleotide sequence existing between these two sequences or between these two sequences and using the nucleotide sequence satisfying the above condition (iii) as a target is used, the inverse occurring in the 22nd intron of the F8 gene, more specifically int22h-1 int22h-3. &lt; / RTI &gt;

According to a specific embodiment of the present invention, the RNA-inducing endonuclease used in the present invention is Cas 9 (CRISPR associated protein 9).

According to a specific embodiment of the present invention, the guiding RNA used in the present invention can be obtained by specifically amplifying the nucleotide sequence of the first sequence of the sequence listing and the nucleotide sequence of the second sequence of the sequence listing, or a complementary sequence thereof It is a guiding RNA that recognizes.

According to the present invention, the RNA-inducing endonuclease and the inducible RNA of the present invention can be used in the form of proteins and transcribed RNAs, respectively, and transformed into vectors carrying DNAs encoding them, respectively, .

According to another aspect of the present invention, there is provided a composition for inversion of blood coagulation factor VIII (F8) gene comprising:

(a) RNA-derived endonuclease or RNA-derived endonuclease encoding nucleotides; And

(b) a nucleotide sequence of 15-25 bp in length contained in the nucleotide sequence of the fourth sequence of the sequence listing, wherein no nucleotide sequence having more than 80% sequence homology is present in the nucleotide sequence of the fourth sequence of the sequence listing A guiding RNA that specifically recognizes a 5'-N (G / A) G-3 'nucleotide linked downstream, or a complementary sequence thereof, or a nucleotide encoding the derived RNA.

According to the present invention, it is possible to calibrate the inversion occurring in the intron 22 of the F8 gene according to the above-described embodiment of the present invention, but it is also possible to use the inducible gene targeting the sequence in the intron 1 of the F8 gene of the sequence By using RNA, it is also possible to correct the inverses originating from intron 1 of the F8 gene.

According to a specific embodiment of the present invention, the derived RNA used in the present invention specifically recognizes the nucleotide sequence of SEQ ID No. 3 or its complementary sequence. Sequence Listing The third sequence is a contiguous nucleotide sequence present in intron homolog 1 (int1h-1) and int1h-2 in the first intron, and is generated between int1h-1 and int1h-2 Can be corrected.

According to a specific embodiment of the present invention, the RNA-inducing endonuclease used in the present invention is Cas 9 (CRISPR associated protein 9).

According to yet another aspect of the present invention, the present invention provides a method for producing an induced pluripotent stem cell in which the inverse of the blood coagulation factor VIII (F8) gene is calibrated, comprising the steps of:

(a) reverse-differentiating somatic cells isolated from patients suffering from type A hemophilia to obtain induced pluripotent stem cells;

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

The induced pluripotent stem cells obtained by repopulating somatic cells isolated from patients suffering from type A haemophilia retain the patient-specific inverted genes, which can be calibrated in vitro by the method of the present invention described above. That is, if an induction RNA that specifically recognizes the inversion site is used after examining the site where the inversion occurs, the inversion is corrected at a certain frequency to obtain a patient-customized induced pluripotent stem cell having the same genotype as the wild type .

As used herein, the term &quot; induced pluripotent stem cell &quot; refers to a cell that has been induced to have pluripotent differentiation potential through artificial reprogramming from differentiated cells, and is also referred to as a degeneration inducing pluripotent stem cell do. IPS cells has substantially the same properties as ES cells, in particular cell appearance, it has a pre multipotential in gene and protein expression pattern is similar, in and to the bit vivo, to form a teratoma (teratoma), Gene Of 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, cattle, sheep, dog, cat, mouse, rabbit and the like, Induced pluripotent stem cells, most specifically induced pluripotent stem cells derived from patients with type A haemophilia.

As used herein, the term &quot; de-differentiation &quot; refers to the epigenetic regression process that renders existing differentiated cells into an undifferentiated state to enable the formation of new differentiated tissues. It is also referred to as a reprogramming process and refers to epigenetic changes ). &Lt; / RTI &gt; According to the purpose of the present invention, the term &quot; infertile &quot; is any process for returning differentiated cells having a differentiation ability of 0% or more to less than 100% to undifferentiated state. For example, And dividing the transformed cells into differentiated cells having 1% differentiation potential.

The degeneration of the present invention can be carried out through various methods known in the art that can restore the differentiation ability of already differentiated cells. For example, OCT4, NANOG, SOX2, LIN28 , KLF4, and c-MYC. &Lt; / RTI &gt;

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., &Lt; RTI ID = 0.0 &gt; And the MCDB series (Ham, RG et al., In Vitro 14: 11 (1978)) can be used, but the present invention is not limited thereto .

As used herein, the term &quot; gene transporter &quot; means an agent for introducing and expressing a desired target gene into a target cell. The ideal gene transducer is harmless to the human body or the cell, it is easy to mass-produce, and it is necessary to be able to efficiently transfer the gene.

As used herein, the term &quot; gene transfer &quot; means that a gene is carried 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. As used herein, the term &quot; operably linked &quot; means a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The regulatory sequence will regulate transcription and / or translation of the other nucleic acid sequences. In the present invention, the promoter bound to the nucleotide sequence of the present invention is preferably capable of regulating transcription of the rilacsin gene by operating in an animal cell, more preferably a mammalian cell, and includes a promoter derived from a mammalian virus And a promoter derived from the genome of a mammalian cell, such as CMV (mammalian cytomegalovirus) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, U6 promoter, HSV tk promoter, RSV promoter , Promoter of human IL-4 gene, promoter of human IL-4 gene, promoter of human lymphotoxin gene, promoter of human IL-2 gene, promoter of human IL-4 gene, promoter of human IL-2 gene, promoter of EF1 alpha promoter, metallothionein promoter, beta -actin promoter, human IL- But are not limited thereto. Most preferably, the promoter used in the present invention is a CMV promoter and / or a U6 promoter.

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

The nucleotide sequence of the present invention can be applied to all gene delivery systems used for conventional animal transformation and is preferably a plasmid, an adenovirus (Lockett LJ, et al., Clin. Cancer Res. 3: 2075-2080 ), Adeno-associated viruses (AAV, Lashford LS., Et al., Gene Therapy Technologies, Applications and Regulations Ed. A. Meager, 1999), retrovirus (Gunzburg WH, et al., Retroviral ( 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)), viruses (Chamber R., et al., Proc. Natl. Act. Sci. USA 92: 1411-1415 ), Liposomes (Methosin in Molecular Biology, Vol. 199, SC Basu and M. Basu (Eds.), Human Press 2002) or niosomes. More specifically, the gene carrier of the present invention is a plasmid.

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

If the gene delivery system in the present invention is a kit inner (naked) or recombinant DNA molecule is a plasmid, microinjection (Capecchi, MR, Cell, 22 :. 479 (1980); and Harland and Weintraub, J. Cell Biol 101: 1094- 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. method (Wong, TK et al, Gene , 10:87 (1980); Nicolau and Sene, Biochim Biophys Acta, 721: .... 185-190 (1982); and Nicolau et al, methods Enzymol, 149 :. 157 (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)), and most specifically, electroporation can be used.

According to another aspect of the present invention, there is provided an induced pluripotent stem cell produced by the method of the present invention.

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

The induced pluripotent stem cells of the present invention are cells that have been corrected for the gene inversion in hemophilia patients and then are differentiated into appropriate somatic cells to be transplanted into a patient and express the corrected gene to be a useful cell therapy composition for hemophilia A, . Accordingly, the composition of the present invention can directly induce differentiation into a target cell from a non -humanized bovine graft, or can be transplanted into a human after completion of differentiation from an in vitro to a target cell.

Specifically, the type A hemophilia to be treated in the present invention is severe A type hemophilia. As used herein, the term &quot; severe hemophilia A &quot; refers to a case where the activity of blood coagulation factor VIII (F8) is less than 1% of normal persons.

As used herein, the term &quot; treatment &quot; includes (a) inhibiting the development of a disease, disorder or condition; (b) relief of the disease, disorder or condition; Or (c) eliminating the disease, disease or condition. The composition of the present invention has a role of suppressing, eliminating or alleviating the development of a symptom that has been induced on the gene by correcting the same gene as the wild type that has been corrected in an individual afflicted with type A hemophilia. Therefore, the composition of the present invention may be a therapeutic composition of hemophilia type A by itself, or may be administered together with other pharmacological components to be used as a therapeutic aid for the disease. Herein, the term &quot; treatment &quot; or &quot; therapeutic agent &quot; includes the meaning of &quot; therapeutic aid &quot;

The term &quot; administering &quot; or &quot; administering &quot; as used herein refers to the administration of a therapeutically effective amount of a composition of the present invention directly to a subject to form the same amount in the body of the subject. Thus, the term &quot; administering &quot; is used to refer to the use of the term &quot; injected &quot; or &quot; transplantation &quot; as it includes injecting or transplanting adult pluripotent stem cells "Is used in the same sense.

A &quot; therapeutically effective amount &quot; of a composition means an amount of extract sufficient to provide a therapeutic or prophylactic effect to the subject to which the composition is to be administered, including a &quot; prophylactically effective amount &quot;. As used herein, the term &quot; subject &quot; includes without limitation humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the object of the present invention is human.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, or a medium, but are not limited thereto.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered parenterally, specifically, 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, . Typical dosages of the pharmaceutical compositions of this invention are 10 &lt; ² &gt; -10 &lt; ¹⁰ &gt; 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 by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, the present invention provides a composition for inducing inversion of blood coagulation factor VIII (F8) gene comprising:

(a) RNA-derived endonuclease or RNA-derived endonuclease encoding nucleotides; And

(b) a guiding RNA that specifically recognizes the following cleotide sequence or a complementary sequence thereof, or a nucleotide encoding the derived RNA:

(I) a nucleotide sequence of SEQ ID NO: 1;

(Ii) the nucleotide sequence of SEQ ID NO: 2; And

(Iii) a 15-25 bp nucleotide sequence present in nucleotides 562,975 bp in length between the above (i) and the above (ii) on the 22nd intron of the F8 gene

(Iii) is at least 80% identical to (i) or (ii) in the 562,975 bp long nucleotide between (i) and (ii) Wherein no nucleotide sequence with sequence homology is present and 5 &apos; -N (G / A) G-3 &apos; nucleotides are linked downstream.

According to another aspect of the present invention, the present invention provides a composition for inducing inversion of blood coagulation factor VIII (F8) gene comprising:

(a) RNA-derived endonuclease or RNA-derived endonuclease encoding nucleotides; And

(b) a nucleotide sequence of 15-25 bp in length contained in the nucleotide sequence of the fourth sequence of the sequence listing, wherein no nucleotide sequence having more than 80% sequence homology is present in the nucleotide sequence of the fourth sequence of the sequence listing A guiding RNA that specifically recognizes a 5'-N (G / A) G-3 'nucleotide linked downstream, or a complementary sequence thereof, or a nucleotide encoding the derived RNA.

Since each configuration used in the present invention has already been described above, the description thereof will be omitted in order to avoid excessive duplication.

In accordance with the present invention, the method of the present invention can induce recurrence or correction of inversions occurring when a patient cell in which an inversion occurs is used as a starting material, but conversely, when using normal cells as a starting material, have. Therefore, by using a cell having a normal genotype as a starting cell, the present invention can be a useful research tool for efficiently obtaining an artificial hemophilia cell.

According to another aspect of the present invention, there is provided a method for inducing inversion of blood coagulation factor VIII (F8) gene comprising introducing a composition for inducing inversion of blood coagulation factor VIII (F8) gene of the present invention into a somatic cell of a normal person &Lt; / RTI &gt;

Since each composition and step used in the present invention has already been described above, the description thereof is omitted in order to avoid excessive overlapping.

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

(a) The present invention relates to a method for inversion of normal blood coagulation factor VIII (F8) gene, a method for inversion of blood coagulation factor VIII (F8) gene inversion, Derived derived pluripotent stem cells.

(b) The method of the present invention efficiently reproduces the inversion of intron 1 and intron 22 of the F8 gene, which accounts for a large number of causes of severe A-type hemophilia, and thereby provides a research tool for investigating the pathogenesis mechanism of type A hemophilia, can be used as a tool.

(c) The inverse-proof inducible pluripotent stem cells prepared by the method of the present invention are useful for restoring the mutant genotype to a wild-type state without restricting the normal gene or protein transfer, And enables the underlying treatment.

FIG. 1 is a graph showing the results of calibration of partial-inverted F8 gene in type-A hemophilia-derived iPSC. Figure la shows that by separating genomic DNA from human dermal fibroblast (WT) and type-A hemophilia patient-derived urine cells (Pa1, Pa2 and Pa3) and performing PCR analysis with appropriate primers (Bagnall et al., 2006) (Left) or 22 (right) inversion. 1B is a schematic diagram showing inversion and recovery of intron 22. Three homologous sites were designated as int22h-1, int22h-2 and int22h-3, respectively. The blue arrowhead indicates the PCR primer. Nuclease target sites near int22h-1 or int22h-3 were expressed in black (RGEN 02) or red (RGEN 03) lightning, respectively. Figure 1C shows the mutation of the nuclease target site in HeLa cells by T7E1 assay. Figure 1d shows the PCR product corresponding to the inversion of the 563-kbp chromosomal segment in HeLa cells. Figure 1e shows the results of PCR analysis confirming inversion calibration in iPSC clones. Figure 1f shows the inflection point junction DNA sequence in inverted-calibrated iPSC clones. Each RGEN target sequence is underlined, the PAM sequence is shown in green, and the dotted line indicates deleted bases. The lower case indicates the inserted sequence and the blue arrow shows the cutting site.
FIG. 2 is a graph showing the results of analyzing characteristics of inverse-corrected iPSC clones. Figure 2a shows a quantitative real-time PCR (qPCR) results for detecting endogenous OCT4, SOX2, LIN28 and NANOG mRNA in the parental cell line and calibration. Expression levels of each gene were normalized by GAPDH . Figure 2b shows in vitro differentiation of inverse-corrected cell lines. The expression of each marker protein is differentiated into ectoderm (Nestin), mesoderm ([alpha] -smooth muscle actin) and endoderm [AFP (alpha-fetoprotein)]. Scale bar, 50 mm. FIG. 2C shows the results of detection of human ESC-specific markers OCT4 and SSEA4 through immunocytochemistry. Scale bar, 100 mm. The karyotype of each iPSC cell line is also indicated. Figure 1d shows F8 gene expression in differentiated cells in intron 1 and 22 invert-corrected iPSC cell lines. Derived from wild-type iPSCs (WT), patient iPSCs (Pa1, Pa2 and Pa3) and inverse-corrected Pa1- (Co-1 and Co-2) or Pa2-iPSCs (Co-1, Co-2 and Co-3) Expression of F8 and mesodermal markers ( Brachyury ) in one cell was measured by RT-PCR (top) and qPCR (bottom). GAPDH expression was used as a loading control. Figure 2e is a chromatogram showing the correct splicing between exons 1 and 2 or exons 22 and 23 in inverted-calibrated iPSC cell lines.
FIG. 3 is a graph showing the correction of the intron 1 inversion in a type-A hemophilia patient-derived iPSC. FIG. 3A shows a schematic diagram of a process in which the intron 1 inversion found in patients with severe hemophilia A is corrected. The blue arrowhead indicates the PCR primer. FIG. 3B shows the mutation frequency of RGEN 01 target site in intron 1 of F8 gene in HeLa cells through T7E1 analysis. FIG. 3B shows a PCR product corresponding to the inverted genotype of HeLa cells. FIG. 3D is a diagram showing the DNA sequences of two intron 1-phase bodies (named int1 homolog 1 and 2) and the inflection point junction. Genomic DNA was isolated from HeLa cells transfected with an RGEN-encoding plasmid, and the reverse PCR-specific bands were isolated and sequenced at the two inflection points. The RGEN target sequence is underlined, the PAM sequence is indicated in green, and the dotted line indicates deleted base. Lower case indicates inserted base. Figure 3e shows the result of genomic DNA PCR analysis of four calibrated clones (Co-1 to Co-4). Genomic DNA was isolated from wild-type iPSC (WT), a positive control for urine-derived cells and inverted or normal genotypes of intron 1 inverted patient (Pa1-U), respectively. Figure 3f shows the DNA sequences of the two homologues (named int1 homolog 1 and 2) and the inflection point junction. Genomic DNA was isolated from iPSCs transformed with an RGEN-encoding plasmid. Restoration-specific PCR bands were isolated and the sequences of the two inflection point junctions were analyzed. The RGEN target sequence is underlined, the PAM sequence is green, and the dashed line is the deleted base.
Figure 4 is a plot showing targeted inversion and recovery in HeLa cells or patient iPSC. Figure 4a shows the DNA sequence of the two inflection point junctions for inversion in HeLa cells. Each RGEN target sequence is underlined, the PAM sequence is green, and the dotted line indicates deleted bases. Lower case indicates inserted base and blue arrow indicates cutting site. 4B and 4C show DNA sequences of the inflection point junctions at the intron 1 (4b) and intron 22 (4c) sites, respectively. RGEN RNP was delivered directly to intron 1 (Pa1-iPSCs) or intron 22 (Pa3-iPSCs) inversion cells via electroporation. Total DNA was isolated from iPSC and sequenced. Each RGEN target sequence is underlined, the PAM sequence is green, and the dotted line indicates deleted bases. Lowercase letters indicate inserted bases, and two blue arrows indicate truncation sites. When the sequence is detected more than once, the number detected is indicated by the number in parentheses.

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

Experimental Method

Analysis of urine-derived iPSC in patients with type A hemophilia according to the present invention was made under the approval of the Research Ethics Review Committee of the Yonsei University (IRB # 4-2012-0028). All volunteers signed a written consent form prior to urine sample donation for human iPSC generation.

Cell culture and transformation

HeLa cells (ATCC, CCL-2) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% FBS (fetal bovine serum), 0.1 mM nonessential amino acid and 1% antibiotic. To induce DSB, 1 X 10 ⁵ HeLa cells were co-transformed with Cas9-encoding plasmid and sgRNA-encoding plasmid (0.5 mg each) using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Human dermal fibroblast-derived wild-type iPSCs (WT-iPSC Epi3 line) (Park et al., 2014) and urine-derived iPSCs purchased from WiCell Inc. were cultured in hESC medium [20% ML of bFGF (basic fibroblast growth factor, PeproTech) in DMEM / F12 (Gibco) supplemented with 1% non-essential amino acid (Invitrogen) and 0.1 mM 2-mercaptoethanol (Sigma) &Lt; / RTI &gt; To induce recovery in urine-derived iPSC, 5 μg Cas9-encoding plasmid and 5 μg sgRNA-encoding plasmid (5 μg each sgRNA for intron 22 inversion) were added to 1 × 10 ⁶ cells using an electroporation system (Neon; Invitrogen) Plasmid) was electroporated.

In order to directly transfer the Cas9 protein into urine-derived iPSC, transformation was performed by slightly altering the method reported in the prior art (Kim et al., 2014). Cas9 protein (15 μg) was mixed with 20 μg of transcribed sgRNA (20 μg of each sgRNA plasmid for intron 22 inversion) and incubated at room temperature for 10 minutes to allow formation of RNP ribonucleoprotein (RNP). RNP was transformed into 2 X 10 ⁵ iPSCs through a micropore system.

Isolation and expansion of urine-derived cells from patients with severe hemophilia A

Urine samples were collected from eleven patients diagnosed with severe type A hemophilia and the diagnosis was clinically confirmed from the Korean Hematology Foundation. Urine-derived cells were isolated by the methods reported in the past (Zhou et al., 2012). In summary, cells were harvested from approximately 100 ml of midstream urine by centrifugation at 400 g for 10 minutes. Cells were washed twice with PBS and incubated in DMEM / Ham's F12 (1: 1) supplemented with 10% (vol / vol) FBS, REGM (obtained from renal epithelial cell growth medium, Single Quot kit (Lonza) 1) medium (Hyclone). After 4 days of culture under these conditions, the cells were cultured in REGM (Lonza) to expand the cells. These cells were divided into 80-90% confluence in a gelatin-coated culture dish. To identify the F8 genotypes, genomic DNA samples isolated from urine-derived patient cells were subjected to PCR analysis using a primer set recognizing appropriate sites near the intron 1 and 22 inversions of the F8 locus (Bagnall et al., 2006 Park et al., 2014).

RGEN composition

Cas9-encoding plasmids were constructed by previously reported methods (Cho et al., 2013). Cas9 protein fused to HA epitope and NLS (nuclear localization signal) was expressed under the CMV promoter. The U6 promoter was used for sgRNA expression (Cho et al., 2014). Purified recombinant Cas9 protein was purchased from ToolGen, Inc. The guide RNA was transcribed in vitro using a MEGA short-script T7 kit (Ambion) in a previously reported method (Kim et al., 2014). The transcribed RNA was purified by phenol: chloroform extraction and quantified by spectrometer.

T7E1 analysis and frequency of targeted inversions

T7E1 assays were performed according to previously reported methods (Kim et al., 2009). Briefly, the PCR amplicon containing the RGEN target region was heated to denature and anneal to form a heteroduplex DNA fragment. The sections were then treated with T7 endonuclease (New England Biolabs) at 37 ° C for 20 minutes to allow cleavage of the mismatch site, and the products were analyzed by agarose gel electrophoresis. The frequency of targeted calibrations at F8 loci was measured by digital PCR analysis in a conventionally reported manner (Kim et al., 2010). Genomic DNA isolated from co-transformed cells with RGEN and sgRNA plasmids was serially diluted using lipofectamine 2000 transfection reagent (Invitrogen) and PCR analysis was performed on the diluted samples. Positive band intercepts at each dilution point were determined and the results were analyzed using the Extreme Limiting Dilution Analysis program (HuandSmyth, 2009).

Identification of RGEN-mediated inverted F8 locus in HeLa cells

To confirm the genetic corrective activity of RGEN designed in the present invention, each RGEN was co-transformed into HeLa cells together with the sgRNA expression plasmid. RGEN activity was measured by T7E1 assay.

Targeted RGEN-mediated calibration at the F8 locus of patient-derived iPSC

iPSCs were cultured in STO cell support layer and treated with type IV collagenase. After washing with PBS, the cells were separated into single cells by the previously reported method (Desbordes et al., 2008). These single cells were mixed with RGEN and sgRNA plasmids and pulsed at 850 volts for 30 minutes. The cells were then seeded on supporting cells and cultured for 10 days. To detect gene inversion at F8 locus, cells extracted from individual colonies were disrupted and subjected to PCR analysis. The primers used were: 1-F1: 5'-AAATCACCCAAGGAAGCACA-3 ', 1-R1: 5 '-TGGCATTAACGTATTACTTGGAGA-3'; 1-F2: 5'-TGCTGAGCTAGCAGGTTTAATG-3 ', 1-R2: 5'-TGCTGAGCTAGCAGGTTTAATG-3'; 22-F1: 5'-TGGGGCTGTGTAAATTTGCT-3 ', 22-R2: 5'-CAAACGTAGCATTACCTGATTGT-3'; 22-F2: 5'-ACAACCAGAGCAGAAATCAATGA-3 ', 22-R2: 5'-TTTCACCACATCCACGCCAA-3'.

Establishment and Characterization of Clonal Cell Groups

To isolate the clones of the calibrated cells, each of the colonies identified by PCR was subjected to the desired genetic modification (inversion genotype correction), which was then separated into single cells and seeded into a new cell support layer. After four passages, several clones (four clones with intron 1 and three clones with intron 22) were selected for sequencing and further experiments were conducted. For sequencing of the inflection point, amplified PCR products were electrophoresed and eluted from the agarose gel.

RNA isolation, RT-PCR and qPCR

Total RNA was purified using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. CDNA was synthesized from total RNA (1 μg) using DiaStar ^™ cDNA synthesis kit (SolGent, Korea). In order to confirm the expression of Factor VIII , Brachyury and GAPDH , PCR was performed using Ex-Taq (Takara) as a template for the synthesized cDNA. For qPCR, SYBRPremix Ex-Taq (Takara) was used according to manufacturer's instructions. In order to amplify F8 mRNA from intron 1 or intron 22 calibration cell lines, a forward primer located in exon 1 (or exon 21 in intron 22 correction cell line) was added to the reverse primer located in exon 3 (or exon 23 in intron 22 calibration cell line) Respectively. The following primer sets were used for RT-PCR or qPCR: GAPDH- F: 5'- CCCCTCAAGGGCATCCTGGGCTA-3 ', GAPDH -R: 5'-GAGGTCCACCACCCTGTTGCTGTA-3'; OCT4 -F: 5'-CCTCACTTCACTGCACTGTA-3 ', OCT4 -R: 5'-CAGGTTTTCTTTCCCTAGCT-3'; LIN28- F: 5'-AGCCATATGGTAGCCTCATGTCCGC-3 ', LIN28- R: 5'-TCAATTCTGTGCCTCCGGGAGCAGGGTAGG-3'; SOX2- F: 5'-TTCACATGTCCCAGCACTACCAGA-3 ', SOX2- R: 5'-TCACATGTGTGAGAGGGGCAGTGTGC-3'; NANOG- F: 5'-TGAACCTCAGCTACAAACAG-3 ', NANOG- R: 5'-TGGTGGTAGGAAGAGTAAAG-3'; F8 -exon 1-F: 5'-CTGCTTTAGTGCCACCAGAAGA-3 ', F8 -exon 3-R: 5'-GACTGACAGGATGGGAAGCC-3'; F8 -exon 21-F: 5'-CCGGATCAATCAATGCCTGGAG-3 ', F8 -exon 23-R: 5'-ATGAGTTGGGTGCAAACGGATG-3'; Brachyury- F: 5'-ATCACAAAGAGATGATGGAGGAA-3 ', Brachyury- R: 5'-GGTGAGTTGTCAGAATAGGTTGG-3').

Production of urine-derived iPSC and in vitro differentiation

IPSCs were generated from urine-derived cells after incubation in sub-3 passages. IPSCs were prepared from urine-derived cells according to the methods previously reported using an episomal reprogram vector or Sendai virus (Invitrogen) (Okita et al., 2011). Seven iPSC colonies, similar in shape to human ES cells, were picked up mechanically and further cultured for characterization. The iPSC is expressed in vitro using three well known methods: (Sugii et al., 2010). Embryoid body (EB) formation was induced by partially isolating iPSC through type IV collagenase (Invitrogen). EBs were transferred to Petri dishes (SPL Lifesciences, Korea) and differentiated for 10 days in hESC medium supplemented with 5% FBS without bFGF. Whether EBs were spontaneously differentiated into cells representing the three germ layers was investigated by immuno staining using appropriate antibodies. In order to differentiate iPSC into mesodermal lineages, the previously reported method was slightly modified (Yoo et al., 2013). Briefly, EBs were transferred to petri dishes and cultured in hESC medium supplemented with 20 ng / mL BMP4 (bone morphogenic protein-4, R & D Systems) and 10 ng / mL actin A (PeproTech) without bFGF. On the third day, EB was plated in a Matrigel-coated dish and further cultured in the above-mentioned medium for 3 days. On the 6th day of differentiation, cells differentiated into mesodermal lineages were collected and examined for F8 gene expression.

Characterization of iPSC

Alkaline phosphatase activity was measured using a leukocyte alkaline phosphatase staining kit (Sigma) according to the manufacturer's instructions. STR (hort tandem repeat) was analyzed to confirm that the iPSC cell line was generated in urine-derived cells. STR loci were amplified from genomic DNA samples isolated from iPSC cell lines and parental cells using the AmpFISTR PCR reaction system (Applied Biosystems). PCR-based STR analysis was performed in Human Pass Inc (Korea). For karyotyping analysis, G-banding analysis for each iPSC cell line derived chromosome was performed in GenDix Inc (Korea). Immunostaining for ES cell markers was performed according to the methods reported previously (Park et al., 2014). DAPI (4 ', 6-diamino-2-phenylindole, Vector Laboratories) was used for nuclear visualization and images were captured and analyzed using an Olympus IX71 microscope or FSX system.

Targeted in-depth sequencing

Genomic DNA segments containing nuclease target sites were amplified using Phusion polymerase (New England Biolabs). Pair-end read sequencing was performed using IlluminaMiSeq for the same amount of PCR amplicon. Excess lead less than end.005% leads to eliminating sequence lead. In the vicinity of the RGEN cleavage site (PAMen3bp upstream), l was considered a mutation induced by RGEN.

Total gene sequencing

DNeasy Tissue kit (Qiagen) was used to purify genomic DNA according to the manufacturer's instructions. Genomic DNA (1 μg) was sectioned using the Covaris system and blunt ends were formed through an End Repair Mix. The fragmented DNA was ligated with an adapter to construct a library. The library was sequenced in Macrogen (Korea) using Illumina HiSeq X Ten Sequencer.

Whole gene sequencing analysis and mutation information extraction

The FASTQ files obtained from the Illumina HiSeq X Ten Sequencer were analyzed in Macrogen via the Isaac workflow of Illumina, Inc (USA). In summary, the FASTQ file read by the fair end was aligned using Isaac Genome Alignment software (Isaac Aligner) according to the reference gene hg19. Then, SNP (single nucleotide polymorphism) and indels were identified as Isaac Variant Caller. Among the many variations, the inventors focused on indel because RGEN hardly induced substitution. The bioinformatic filter was applied to exclude indel extracted from public databases and indel extracted from other genes. Next, the RGEN target region was compared with the wild type locus corresponding to the indel position. The 31-106 indel sites contained 5'-N (G / A) G-3'PAM sequences, each with an on-target sequence and a minimum of 12 nucleotide matches. Finally, targeted deep sequencing was performed on 10 indel sites after excluding sites with repeat sequences.

Inspection of potential off-target areas

To investigate the presence of nuclease-induced indels in a number of potential off-target sites in each gene sequence, Cas-OFFinder was used to determine whether the on-target region and up to 8 nucleotides were different, All potential off-target sites that differed in RNA bulge and up to two nucleotides were identified (Bae et al., 2014). An internal computer program was used to generate a conservative CIGAR string of 20% of the entire CIGAR string at each potential off-target site. Next, we compared the conservative CIGAR string of a number of potential off-target sites with the CIGAR string of the reference sequence. As a result, 83-348 potential off-target sites were obtained. Finally, targeted deep sequencing was performed on four indel sites that were observed only in the independent clones and did not have repeat sequences around the target site.

Experiment result

First, we analyzed genotypes of eleven patients with severe A type hemophilia who were not related to each other, and identified one patient with int1 inversion and two patients with int22 inversion. Two patients (named "Pa2" and "Pa3") with one int1 inversion (named "Pa1") and an int22 inversion were selected for the experiment (FIG. Each iPSC was established by introducing four Yamanaka factors into the intestinal epithelial cells via episomal vector or Sendai virus to obtain fibroblasts of these patients with hemorrhagic disease and avoid invasive biopsy. At the same time, we investigated whether RGEN composed of Cas9 protein and sgRNA (small guide RNA) had activity to induce or repair these inversions in wild type HeLa cells and patient iPSC. RGEN 01 was designed to target sites within int1h (Figure 3a). RGEN 01 was very active and induced small insertions and deletions (indels) at a frequency of 34% at the target site in int1h (Fig. 3b). Furthermore, RGEN 01 induces a 140-kbp chromosomal segmental inversion of HeLa cells as shown in the inverse-specific PCR (Figure 3c). As a result of digital PCR analysis, the frequency of inversion was measured as 2.2% -3.1% (Kim et al., 2010; Lee et al., 2010).

The present inventors analyzed the DNA sequence of the reverse-specific PCR amplicons and found that indel was induced at two inverted breakpoint junctions (FIG. 3D). Based on this high frequency, we co-transform plasmids encoding Cas9 protein and sgRNA to Pa1-derived iPSCs (Pa1-iPSCs) and analyzed iPSC colonies using PCR. Eight colonies of 120 colonies (not necessarily from one cell) (6.7%) produced positive PCR bands on the agarose gel.

Four colonies were further cultured to obtain a single cell-derived clone. These clones generated PCR amplicons corresponding to the int1h-1 and int1h-2 sites, indicating that the inversion in the 140-kbp chromosomal segment of Pa1 cells was restored (Fig. 3e).

On the other hand, these PCR amplicons were not generated from Pa1 iPSCs or urinary cells. We sequenced the PCR amplicon and observed that indel was not induced at the target site of the three clones. In another clone, 3-bp deletion was detected at the target site (Figure 3f).

The present inventors have also focused on other int22h regions. In addition to restoring the desired 600-kbp segmental inversion, two RGENs targeting the outer portion of the homologue were used (Figure 1b) to rule out the possibility of unwanted deletions or inversions involving two or three int22 homologues. This strategy also allows for the detection of restoration using appropriate primers. The present inventors designed two RGENs (RGEN 02 and RGEN 03) to target sites near int22h-1 and int22h-3 and tested for nuclease activity in HeLa cells through T7E1 (T7 endonuclease I) analysis Respectively. These RGENs were highly active and induced indel at the target site at 44% or 32% frequency (Fig. 1C). Next, the inversion of the 563-kbp chromosome segment between the two target sites was detected by PCR. Transgenic-specific PCR amplicons were not generated when RGEN 02 or RGEN 03 alone were transfected into HeLa cells. On the other hand, co-transformation of these RGEN plasmids produced two inverted-specific PCR amplicons (Fig. 1d). The frequency of inversion is 1.5% -2.2% (Table 1).

Targeted inversion frequency in HeLa cells Genomic DNA amount (copy number per gene) frequency (%) p value
(Fit) 1 ng
(300) 333 pg
(100) 100 pg
(30) 33 pg
(10) 10 pg
(3) Estimate Upper and lower limits ^* int1h1 16/16 16/16 10/16 3/16 0/16 3.1 (2.0-4.7) 0.04 int1h2 16/16 15/16 7/16 3/16 0/16 2.2 (1.4-3.3) 0.24 int22
(junction1) 16/16 13/16 8/16 6/16 0/16 2.2 (1.4-3.3) 0.43 int22
(junction3) 16/16 13/16 6/16 0/16 - 1.5 (1.0-2.3) 0.13

^{* The} upper and lower limits represent 95% confidence intervals.

The DNA sequences of the PCR amplicons showed that the two DSGs induced by two RGENs were restored by non-homologous end joining (error-frequent) by observing that indel was mostly accompanied by two inverted inflection points. (Fig. 4A). HeLa cells are wild-type in the direction of the F8 exon. In Pa2 and Pa3 cells, F8 exons 1-22 were inverted. However, these two RGEN target regions are still conserved, and large amounts of chromosomal segments can be recovered.

Next, RGEN 02 and 03 were transformed into Pa2 iPSC through electroporation, 135 colonies were isolated, and PCR analysis of their genomic DNA was performed. Five colonies (3.7%) produced PCR amplicons corresponding to reverse-correction (i. E., Recovery). This PCR product was not obtained in Pa2 iPSCs or wild-type iPSC (Fig. 1e). These colonies were expanded to isolate three independent single cell-derived clones. DNA sequences at the junctions of two inverted inflection points were examined to see if the 563-kbp chromosome segment between the two RGEN sites was restored (Fig. 1F). As a result, indel, which is characteristic of error - frequent NHEJ as in HeLa cells, was observed at the two inflection point junctions of these inverse - corrected iPSCs.

We investigated whether inverted-calibrated iPSCs retain pluripotency. First, the inversion was confirmed that the corrected Pa1 (int1h inversion) and Pa2 (int22h inversion) results of testing the expression of stem cell markers in iPSC OCT4, SOX2, LIN28 and NANOG is actively transferred (Fig. 2a). In addition, these inversely-calibrated iPSCs successfully differentiated into three primary glands (Fig. 2b) and further showed normal karyotypes (Fig. 2c). Taken together, it can be seen that overall chromosomal recovery by RGEN has no negative impact on the versatility of patient-derived iPSCs.

Mesodermal-derived epithelial cells are a major source of F8 gene expression (Shahani et al., 2010). We differentiated the patient iPSC and inverse-corrected patient iPSC into mesodermal lobe and then measured the level of F8 mRNA by RT-PCR. As expected, the detection of PCR bands corresponding to F8 exons 1 and 2 in the cells differentiated in Pa1-iPSC revealed that F8 was not expressed in the patient-derived cells (Fig. 2d). On the other hand, PCR bands corresponding to these exons were detected in cells differentiated from wild-type iPSC or two inverted-calibrated Pa1-iPSCs (Co-1 and Co-2). Similarly, PCR amplicons corresponding to F8 exons 22 and 23 were not detected in the cells differentiated in Pa2- and Pa3-iPSC, but were detected in cells differentiated in three inverted-calibrated iPSCs (Fig. 2d).

Sequence analysis confirmed that exons 1 and 2 or exons 22 and 23 were correctly spliced in cells differentiated from inverted-calibrated iPSCs (FIG. 2e). These results show that the F8 gene has been corrected in patient iPSC with intron 1 and 22 inversions.

To prevent unwanted insertion of the plasmid fragments at the RGEN on-target and off-target sites, two patient iPSCs (Pa1 and Pa3) with intron 1 or 22 inversion were used to express the purified recombinant Cas9 protein after expression in E. coli and (RGN ribonucleoprotein, RNP) transcribed in vitro.

PCR and seaweed sequencing were used to confirm the recovery of the 140-kbp or 563-kbp chromosomal segments restoring the genetic function of the F8 gene (Figures 4b and 4c). Unlike the plasmid, RNP immediately cleaves the target DNA of the chromosome immediately after transfection and rapidly degrades in the cell (Kim et al., 2014), thus sacrificing the genetic corrective activity of the on-target site through transduction of RGEN RNP It is possible to reduce the off-target effect.

RGEN can induce the same off-target mutation as the on-target region (Cho et al., 2014; Cradick et al., 2013; Fu et al., 2013; Hsu et al., 2013; al., 2013). We used targeted deep sequencing and whole genome sequencing (WGS) to investigate whether RGEN used in the present invention leaves secondary damage other than inversion of inverse in patient iPSC. First, using a web-based program, Cas-OFFinder ( www.rgenome.net , Bae et al. 2014), three RGEN on-target regions in the human gene and three potential nucleotide- Respectively. A total of 12, 6 and 14 sites were found in RGEN 01, RGEN 02 and RGEN 03, respectively. Targeted in-depth sequencing was used to verify that indel was not induced at these sites in the inverted-calibrated iPSC (Table 2).

Analysis of potential off-target areas

Next, WGS was performed on genomic DNA isolated from patients (Pa1 and Pa2) iPSC and each inverse-corrected iPSC. First, Isaac, a mutation information extraction program, was used to identify indel based on the hg19 standard gene. Indel extracted from patient genes with indel, int1h or int22h inversion and other inverted-corrected genes listed in the public database by applying bioinformatics filters, and indel with homo-polymer or repeat sequences due to sequencing errors I excluded. As a result, 9,848-13,707 indels unique to each inverse-corrected gene sequence were obtained. Next, the RGEN target region was compared with the wild type locus corresponding to the indel position. Only 31-106 indel sites had 5'-N (G / A) G-3'PAM sequences, with at least 12 nucleotide matches with each on-target sequence (Table 2). We then examined the DNA sequences corresponding to these indel in two inverted-corrected genes. No indel was identified as targeted deep sequencing. Next, all potential off-target sites that differ from the on-target region by up to eight nucleotides, or from five base DNA or RNA bulges to up to two nucleotides, Respectively. Subsequently, the sequences of the 10 sequences from the thousands of potential off-target sites were compared with the standard sequences (Table 2), and the off-target indel was not identified. These results show that the three RGENs used in the present invention do not induce off-target mutations in inverse-corrected patient iPSC. In summary, we used RGEN in patient-derived iPSC to restore two large-scale chromosomal inversions that accounted for half of severe hemophilia A, thereby allowing differentiated cells in inverse-corrected iPSC to express F8 Gene expression was observed. Targeted in-depth sequencing and WGS analysis confirmed that off-target mutations were not induced in inverted-calibrated iPSCs. This is the first demonstration that large gene rearrangements such as chromosome inversion can be corrected in patient iPSC using RGEN or other gene scissors. Chromosome inversion is also associated with other genetic disorders such as Hunter syndrome (Bondeson et al., 1995) and cancer (Nikiforova et al., 2000). Targeted gene rearrangement in iPSC using RGEN is a method of studying their function by allowing structural modification of gene and gene and cell therapy method of various genetic diseases caused by extensive chromosome rearrangement including A type hemophilia Can be usefully used.

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> Endonuclease for Targeting Blood Coagulation Factor 8 and Use          Thereof <130> PN140652 <160> 29 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> homo sapiens <400> 1 cccgtataac tcttaaacac 20 <210> 2 <211> 20 <212> DNA <213> homo sapiens <400> 2 tggtaagagt accggtggga 20 <210> 3 <211> 20 <212> DNA <213> homo sapiens <400> 3 ggtccccggg gttgtgcccc 20 <210> 4 <211> 1041 <212> DNA <213> homo sapiens <400> 4 cagttgtcag tatgtgaaca atgttggaaa catgttattt tgtctgattt cctttgggag 60 aattcattgc cagctataaa tctgtggaaa cgctgccaca caatcttagc acacaagatt 120 ggcagaaaat cgcttagaaa cactgaaaac atgtgacaaa gtgctttccg tgaaaagggt 180 ggatgcgaag cagtaaggac ccccttcatg aagcacgcgg tcaccctcca gccaccagaa 240 ccagaggaac gctgtggtaa ctgagggaaa acgcatctag gcacacgtca cggtggcacc 300 ttccagcagg tccccggggt tgtgcccctg gagctctgac aaagagtgtg gcccggaaat 360 gtgatgtttg gactgcaagt tttggtgaga aataacaatg catcaggttg cagacaaagc 420 agaccctgct ctgtgcgttt atgggagccg cgcccaacag gaaccacagg gaatgatcga 480 aaggagaggg acggacacaa acagacacac cagagagagg ttctcaggag gaggctctgt 540 ggcctccaga ccacgtcaaa gccaaggcag aaaggatgaa ctgaggaaag gaggaaaatt 600 tccccttaag gaaggtaaaa tccagaaggg atccctaaaa tggtgagcag tttaaaccta 660 gcagttttgc attaattcac ataaagtata atgaaaactg ttggacacac aaggagagac 720 tggccaatct atagtcacag aggaagacct tcacacccct tcacaggatc tcgcagcaga 780 ttggctgaaa agtctccttg aaactgcaga cctctctcaa ggagacccac tgagttgggc 840 aaaggtgggg ccgacttaat tcttgcctcc ctgctctccc acgtagccct gcatttcact 900 ccattccagg gtttctggga cacccgagaa agcacgtagt ccagggagca cgtctgccaa 960 ctgaaggcct tgacaaatga ctttctgtac tggctgaggg ccagggccca gcgtactgat 1020 aaggaagctc ttccagaaaa a 1041 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 1-F1 <400> 5 aaatcaccca aggaagcaca 20 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 1-R1 <400> 6 tggcattaac gtattacttg gaga 24 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 1-F2 <400> 7 tgctgagcta gcaggtttaa tg 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> 1-R2 <400> 8 tgctgagcta gcaggtttaa tg 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 22-F1 <400> 9 tggggctgtg taaatttgct 20 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 22-R2 <400> 10 caaacgtagc attacctgat tgt 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 22-F2 <400> 11 acaaccagag cagaaatcaa tga 23 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 22-R2 <400> 12 tttcaccaca tccacgccaa 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F <400> 13 cccctcaagg gcatcctggg cta 23 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R <400> 14 gaggtccacc accctgttgc tgta 24 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OCT4-F <400> 15 cctcacttca ctgcactgta 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> OCT4-R <400> 16 caggttttct ttccctagct 20 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> LIN28-F <400> 17 agccatatgg tagcctcatg tccgc 25 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LIN28-R <400> 18 tcaattctgt gcctccggga gcagggtagg 30 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SOX2-F <400> 19 ttcacatgtc ccagcactac caga 24 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> SOX2-R <400> 20 tcacatgtgt gagaggggca gtgtgc 26 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NANOG-F <400> 21 tgaacctcag ctacaaacag 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> NANOG-R <400> 22 tggtggtagg aagagtaaag 20 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F8-exon1-F <400> 23 ctgctttagt gccaccagaa ga 22 <210> 24 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F8-exon3-R <400> 24 ctgctttagt gccaccagaa ga 22 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F8-exon3-R <400> 25 gactgacagg atgggaagcc 20 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F8-exon21-F <400> 26 ccggatcaat caatgcctgg ag 22 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F8-exon23-R <400> 27 atgagttggg tgcaaacgga tg 22 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Brachyury-F <400> 28 atcacaaaga gatgatggag gaa 23 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Brachyury-R <400> 29 ggtgagttgt cagaataggt tgg 23

Claims (16)

Composition for inversion of in vitro or in vitro coagulation factor VIII (F8) gene comprising:
(a) RNA-derived endonuclease or RNA-derived endonuclease encoding nucleotides; And
(b) a guiding RNA that specifically recognizes the following nucleotide sequence or a complementary sequence thereof, or a nucleotide encoding the derived RNA:
(I) a nucleotide sequence of SEQ ID NO: 1;
(Ii) the nucleotide sequence of SEQ ID NO: 2; And
(Iii) a 15-25 bp nucleotide sequence present in nucleotides 562,975 bp in length between the above (i) and the above (ii) on the 22nd intron of the F8 gene
(Iii) is at least 80% identical to (i) or (ii) in the 562,975 bp long nucleotide between (i) and (ii) Wherein no nucleotide sequence with sequence homology is present and 5 &apos; -N (G / A) G-3 &apos; nucleotides are linked downstream.
The composition of claim 1, wherein the RNA-derived endonuclease is Cas 9 (CRISPR associated protein 9).
The method according to claim 1, wherein the guiding RNA comprises a nucleotide sequence selected from the group consisting of a nucleotide sequence of the first sequence of the sequence listing and a nucleotide sequence of the second sequence of the sequence listing or a guiding RNA ). &Lt; / RTI &gt;
2. The composition of claim 1, wherein the composition calibrates the inverse occurring in intron 22 of the blood coagulation factor VIII (F8) gene.
Composition for inversion of in vitro or in vitro coagulation factor VIII (F8) gene comprising:
(a) RNA-derived endonuclease or RNA-derived endonuclease encoding nucleotides; And
(b) a 15-25 bp long nucleotide sequence contained within the nucleotide sequence of the fourth sequence of SEQ ID NO: 4, wherein the nucleotide sequence of the fourth sequence has a sequence homology of at least 80% with the sequence of the fourth sequence (G / A) G-3 &apos; nucleotide linked downstream of the 5'-N (G / A) G-3 &apos; nucleotide or a complementary sequence thereof, Nucleotides encoding RNA.
6. The composition of claim 5, wherein said derived RNA specifically recognizes a nucleotide sequence of SEQ ID NO: 3 or a complementary sequence thereof.
6. The composition of claim 5, wherein the RNA-derived endonuclease is Cas9 (CRISPR associated protein 9).
6. The composition according to claim 5, wherein the composition corrects the inverse occurring in intron 1 of the blood coagulation factor VIII (F8) gene.
A method for producing an induced pluripotent stem cell in which the inverse of the blood coagulation factor VIII (F8) gene comprising the following steps is calibrated:
(a) reverse-differentiating somatic cells isolated from patients suffering from type A hemophilia to obtain induced pluripotent stem cells;
(b) contacting the induced pluripotent stem cell with the composition of any one of claims 1 to 7 or transforming the gene carrier with the inserted composition.
[9] The method of claim 9, wherein the step (a) comprises transforming somatic cells isolated from the patient with type A hemophilia by one or more genes selected from the group consisting of OCT4, NANOG, SOX2, LIN28, KLF4 and c-MYC Lt; / RTI &gt;
An induced pluripotent stem cell produced by the method of claim 9.
A composition for treating type A hemophilia comprising the induced pluripotent stem cell of claim 11 as an active ingredient.
Composition for inducing in vitro or in vitro coagulation factor VIII (F8) gene inversion comprising:
(a) RNA-derived endonuclease or RNA-derived endonuclease encoding nucleotides; And
(b) a guiding RNA that specifically recognizes the following nucleotide sequence or a complementary sequence thereof, or a nucleotide encoding the derived RNA:
(I) a nucleotide sequence of SEQ ID NO: 1;
(Ii) the nucleotide sequence of SEQ ID NO: 2; And
(Iii) a 15-25 bp nucleotide sequence present in nucleotides 562,975 bp in length between the above (i) and the above (ii) on the 22nd intron of the F8 gene
(Iii) is at least 80% identical to (i) or (ii) in the 562,975 bp long nucleotide between (i) and (ii) Wherein no nucleotide sequence with sequence homology is present and 5 &apos; -N (G / A) G-3 &apos; nucleotides are linked downstream.
Composition for inducing in vitro or in vitro coagulation factor VIII (F8) gene inversion comprising:
(a) RNA-derived endonuclease or RNA-derived endonuclease encoding nucleotides; And
(b) a 15-25 bp long nucleotide sequence contained within the nucleotide sequence of the fourth sequence of SEQ ID NO: 4, wherein the nucleotide sequence of the fourth sequence has a sequence homology of at least 80% with the sequence of the fourth sequence (G / A) G-3 &apos; nucleotide linked downstream of the 5'-N (G / A) G-3 &apos; nucleotide or a complementary sequence thereof, Nucleotides encoding RNA.
15. The composition of claim 14, wherein the derived RNA specifically recognizes a nucleotide sequence of SEQ ID NO: 3 or a complementary sequence thereof.
A method for inducing inversion of the blood coagulation factor VIII (F8) gene, which comprises the step of introducing the composition of any one of claims 13 to 15 into a somatic cell isolated from a normal person.

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