JP7249620B2 - nucleic acid binding factor - Google Patents

Description

The present invention relates to nucleic acid binding factors and the like.

Artificial transcription factors mainly consist of a DNA-binding domain and a transcription-inducing (or repressing) domain. Although various artificial transcription factors have been developed so far, there are not so many artificial transcription factors that are single proteins, have no repeated sequences, and act with high specificity to target sequences.

The most commonly used transcription factors, which use the tet repressor protein domain (TetR) as the DNA-binding domain, dimerize and primarily recognize and bind sequences of 12 bp. Since TetR can regulate binding to DNA by tetracycline and doxycycline, this point is useful in the field of experimental science. Difficult to use in humans.

There are also multiple DNA-binding domains that could be used for artificial transcription factors. First, inactivated dCAS9 (inactivated Cas9) can become the DNA-binding domain of an artificial transcription factor that can bind to the target sequence with high specificity due to the presence of gRNA, but the molecular weight of dCas9 is large and Since it is necessary to express gRNA separately, it is inconvenient as an artificial transcription factor for expressing a specific gene. Secondly, TALE can also be an artificial transcription factor with high sequence characteristics, but since it consists of about 20 repeated sequences, it is difficult to use because it is impossible to confirm the gene sequence by Sanger sequencing and to artificially synthesize the gene. In fact, it is known that lentivirus, which is used to introduce genes into therapeutic cells, significantly reduces the efficiency of viral infection if the size of the loaded gene is large. It is known that there is a limit to the size of a gene that can be loaded into an associated virus. Therefore, smaller genes are desirable and more versatile for use in creating therapeutic cells. Third, synthetic zinc finger proteins can bind to specific DNA sequences, but not with high avidity. Fourthly, an artificial transcription factor created using the DNA-binding domain of the yeast-derived transcription factor GAL4 is also frequently used experimentally, but its recognition sequence is about 7 bp and its specificity is considerably low.

In recent years, the use of meganucleases such as SceI as artificial transcription factors has been reported (Patent Document 1). However, the use of a meganuclease derived from the subphylum Chahuangtake as an artificial transcription factor has not yet been reported.

Special Table 2011-519558

An object of the present invention is to provide a nucleic acid binding factor that can be used as an artificial transcription factor.

As a result of intensive research, the present inventors have found that the above problems can be solved by a nucleic acid-binding factor comprising a polypeptide containing a DNA-binding domain of an inactive Chawanthalpus-derived meganuclease. As a result of further research based on this knowledge, the present invention was completed.

That is, the present invention includes the following aspects.

Section 1. A nucleic acid binding factor comprising a polypeptide comprising a DNA-binding domain of an inactive Pseudomonas meganuclease.

Section 2. (A) said polypeptide further comprises a functional domain; and/or (B) apart from said polypeptide further comprises a functional substance,
Item 1. The nucleic acid binding factor according to item 1.

Item 3. (A) The nucleic acid binding agent of item 1 or 2, wherein the polypeptide further comprises a functional domain.

Section 4. 4. Any one of items 1 to 3, wherein the meganuclease derived from the subphylum Chahuangtake is at least one selected from the group consisting of I-OnuI, I-AaBMI, I-PanMI, I-CpaMI, and I-LtrWI. A nucleic acid binding factor as described.

Item 5. Item 5. The nucleic acid-binding factor according to any one of items 1 to 4, wherein the meganuclease derived from the subphylum Chahuangtake is at least one selected from the group consisting of I-OnuI, I-AaBMI, and I-PanMI.

Item 6. The functional domain comprises transcription promoting activity, transcription repressing activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenyl activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, DNA repair activity, DNA editing activity, DNA damage activity, deamination activity activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, nuclease activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity.

Item 7. Item 3. The nucleic acid binding factor according to item 2, wherein the functional substance is at least one selected from the group consisting of a labeling substance and a carrier.

Item 8. Item 8. The nucleic acid binding factor according to any one of Items 1 to 7, which is a transcription factor.

Item 9. Item 9. The nucleic acid binding factor according to any one of Items 1 to 8, wherein the DNA binding domain is a homo DNA binding domain or a chimeric DNA binding domain.

Item 10. A polynucleotide encoding a polypeptide comprising the nucleic acid binding factor of any one of Items 1 to 9.

Item 11. 11. The polynucleotide of Paragraph 10, comprising an expression cassette for said polypeptide.

Item 12. A cell comprising the polynucleotide of Item 10 or 11.

Item 13. A reagent comprising at least one selected from the group consisting of the nucleic acid binding factor of any one of Items 1 to 9, the polynucleotide of Item 10 or 11, and the cell of Item 12.

Item 14. Item 14. The reagent of Item 13, for binding said nucleic acid binding agent to an exogenous nucleic acid.

Item 15. 15. The reagent of Paragraph 14, wherein said exogenous nucleic acid comprises a transcription control sequence.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a nucleic acid binding factor that can be used as an artificial transcription factor. Since this nucleic acid-binding factor has excellent nucleic acid-binding ability, it can be used in various forms other than as a transcription factor (for example, reagents such as carrier reagents for nucleic acid purification and nucleic acid visualization reagents). When used as a transcription factor, for example, the intracellular transcription factor domain is released by placing it in the intracellular domain of a transmembrane protein (for example, by signaling (contact with a specific cell such as a cancer cell) to release a specific gene. It can be used as a form designed to control expression).

A shows an example of an artificial transcription factor produced in Examples. It consists of HA tag sequence, nuclear localization signal (NLS), inactive meganuclease, transcription control domain and proteolytic sequence from the N-terminus. B shows an example of a reporter made in Examples. By placing a CMV mini-promoter downstream of a sequence containing one and seven meganuclease target sequences and placing a reporter gene downstream thereof, binding of an artificial transcription factor using an inactivated meganuclease depends on the binding of the reporter gene. is expressed. In HEK293T cells, reporter plasmids with 7 recognition sequences for meganucleases I-AaBMI, I-CpaMI, I-LtrWI, I-OnuI, and I-SceI were expressed alone. The result of investigating the reporter transcription activity when the expression plasmid of the artificial transcription factor composed of the activated form was co-expressed is shown. The vertical axis indicates the transcription induction ratio, and the horizontal axis indicates the type of meganuclease employed and the presence or absence of an artificial transcription factor expression plasmid. In HEK293T cells, a reporter plasmid with seven meganuclease I-DmoI recognition sequences was expressed alone, and an artificial transcription factor expression plasmid consisting of inactivated meganuclease I-DmoI was co-expressed. 2 shows the results of investigation of reporter transcription activity in the case. The vertical axis indicates the transcription induction ratio, and the horizontal axis indicates the presence or absence of the artificial transcription factor expression plasmid. In HEK293T cells, reporter plasmids with one recognition sequence for the meganucleases I-OnuI and I-PanMI were expressed alone. The results of examining reporter transcription activity when co-expressed are shown. The vertical axis indicates the transcription induction ratio, and the horizontal axis indicates the type of meganuclease employed and the presence or absence of an artificial transcription factor expression plasmid. Fig. 3 shows the results of investigating reporter transcription activity when lymphoma cells (Raji cells) were added to T cells introduced with chimeric Notch receptors containing artificial transcription factors. The vertical axis indicates the transcription induction ratio, and the horizontal axis indicates the type of artificial transcription factor.

(1). Definitions As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

As used herein, "conservative substitution" means that an amino acid residue is replaced with an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains such as lysine, arginine, and histidine correspond to conservative substitutions. In addition, amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine and cysteine; alanine, valine, leucine, isoleucine, amino acid residues with nonpolar side chains such as proline, phenylalanine, methionine, tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, isoleucine; and aromatic side chains, such as tyrosine, phenylalanine, tryptophan, histidine. Substitutions between amino acid residues are also conservative substitutions.

As used herein, nucleic acids (eg, nucleotides such as DNA and RNA) may be subjected to known chemical modifications as exemplified below. Substitution of the phosphate residue of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, phosphorodithionate, etc. to prevent degradation by hydrolases such as nucleases can be done. In addition, the hydroxyl group at the 2nd position of the sugar (ribose) of each ribonucleotide is -OR (R is, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base moiety (pyrimidine, purine) may be chemically modified, for example, introduction of a methyl group or cationic functional group to the 5-position of the pyrimidine base, or substitution of the carbonyl group at the 2-position with thiocarbonyl. is mentioned. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety has been modified with biotin, an amino group, a lower alkylamine group, an acetyl group, or the like. Also included are BNA (LNA), etc., in which the conformation of the sugar portion is fixed to the N-type by bridging the 2' oxygen and 4' carbon of the sugar portion of the nucleotide.

(2). Nucleic acid-binding factor The present invention, in one aspect thereof, comprises a polypeptide comprising a DNA-binding domain of an inactive Chahuangtan-derived meganuclease (herein, also referred to as the "polypeptide of the present invention"). , relates to a nucleic acid binding factor (also referred to herein as "the nucleic acid binding factor of the present invention"). This will be explained below.

The Pezizomycotina-derived meganuclease is not particularly limited as long as it is derived from a species belonging to the Pezizomycotina subphylum (in other words, it is endogenously possessed by the wild type of the species).

Among the subphylum Chawantaceae, preferred are the subphylum Dermatophyta and the class Throccopharyngeal, more preferably are the subphyleus Dymphomacidae and the class Throccopharyngeal, and more preferably the order Ophiostomyces, the order Asteroides, the order Dymphomacidae, the order Diaporte, and the like. More preferably, the families Ophiostomataceae, Helotiaceae, Lasiosphaeriaceae, Cryphonectriaceae, and the like, and particularly preferably the genera Ophiostoma, Ascocalyx, Podospora, Cryphonectria, and Leptographium.

Specific examples of meganucleases derived from the subphylum Chahuangtake include, for example, I-OnuI (Ophiostoma novo-ulmi), I-AaBMI (Ascocalyx abietina), I-PanMI (Podospora anserina), I-CpaMI (Cryphonectria parasitica), I- LtrWI (Leptographium truncatum), I-AniI (Aspergillus nidulans), I-AstI (Annulohypoxylon stygium), I-CcaI (Ceratocystis cacaofunesta), I-CcaII (Ceratocystis cacaofunesta), I-CkaMI (Cordyceps kanzashiana), I-CpaMI ( Cryphonectria parasitica), I-CpaMIIP (Cryphonectria parasitica), I-CraMI (Cordyceps ramosopulvinata), I-GpeMI (Grosmannia penicillata), I-GpiI (Grosmannia piceaperda), I-GzeI (Gibberella zeae), I-GzeII (Gibberella zeae) ), I-GzeMIIIP (Gibberella zeae), I-HjeMII (Hypocrea jecorina), I-LtrII (Leptographium truncatum), I-NcrMIP (Neurospora crassa), I-OmiI (Ophiostoma minus rns), I-OmiII (Ophiostoma minus rns ), I-OsoMI (Ophiocordyceps sobolifera), I-OsoMII (Ophiocordyceps sobolifera), I-OsoMIIIP (Ophiocordyceps sobolifera), I-OsoMIVP (Ophiocordyceps sobolifera), I-PanMIIP (Podospora anserina), I-PanMIIP (Podospora anserina), I-PnoMI (Phaeosphaeria nodorum), I-SmaMI (Sordaria) and the like. Among these, I-OnuI, I-AaBMI, I-PanMI, I-CpaMI, I-OnuI, I-AaBMI, I-PanMI, I-CpaMI, and I- LtrWI. Among these, I-OnuI, I-AaBMI and I-PanMI are particularly preferred, and I-OnuI is more preferred.

The amino acid sequence of the meganuclease derived from the subphylum Chawanmuta can be determined based on publicly known information. For example, the wild-type amino acid sequence for I-OnuI includes SEQ ID NO: 1, the wild-type amino acid sequence for I-AaBMI includes SEQ ID NO: 2, and the wild-type amino acid sequence for I-PanMI includes SEQ ID NO: 3. wild-type amino acid sequences for I-CpaMI include SEQ ID NO:4 and wild-type amino acid sequences for I-LtrWI include SEQ ID NO:5.

"Inactive" indicates a state in which DNA cleavage activity is inactivated. The meganuclease from the subphylum Chawangtake is a monomeric meganuclease, and its structure is clearly divided into two domains, the N-terminal domain and the C-terminal domain, each of which has DNA binding ability and nickase (DNA cleavage) ability. have activity. To inactivate the DNA-cleaving activity, it is necessary to introduce mutations into both the two active centers in the N-terminal and C-terminal domains. This mutation site can be appropriately set according to known information. Specifically, for example, mutation can be introduced into each of the LAGLIDADG motifs, which are the catalytic centers in the N-terminal domain and the C-terminal domain. In particular, it is preferable to replace D (Asp) or E (Glu) on the C-terminal side of the motif with an amino acid other than D or E. In addition to the catalytic center amino acids, meganucleases can also be inactivated by introducing mutations into the metal ion binding site, a structure called BASIC POCKET.

Amino acid sequences of the inactive Chawanguthu subphylum-derived meganucleases include, for example, SEQ ID NO: 6 as an amino acid sequence of inactive I-OnuI, and SEQ ID NO: 7 as an amino acid sequence of inactive I-AaBMI. The amino acid sequence for I-PanMI includes SEQ ID NO:8, the amino acid sequence for inactive I-CpaMI includes SEQ ID NO:9, and the amino acid sequence for inactive I-LtrWI includes SEQ ID NO:10.

An inactive Pseudomonas meganuclease does not significantly impair its DNA binding capacity (e.g., does not reduce DNA binding capacity to 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less). , and the inactivated state of the DNA cleavage activity is not remarkably restored (for example, the DNA cleavage activity is 10% or less, 5% or less, 2% or less, 1% or less, 0.1% of the DNA cleavage activity before inactivation % or less), mutated meganucleases (mutated meganucleases) are also included. DNA-binding ability and DNA-cleaving activity can be measured according to known test methods.

The mutated meganuclease is, for example, an amino acid sequence of an inactive Chawanthalpus-derived meganuclease (unmutated meganuclease) in which no mutations other than mutations for inactivation have been introduced (unmutated meganuclease) and 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more). Mutations include, for example, substitutions, deletions, additions, insertions, etc., preferably substitutions, and more preferably conservative substitutions. The number of mutated amino acids is preferably 1 or more, more preferably 1-8, still more preferably 1-4, and even more preferably 1.

A preferred embodiment of the inactive Pseudomonas meganuclease is the protein according to (a) or (b) below:
(a) a protein consisting of an amino acid sequence in which an inactivating mutation is introduced in the amino acid sequence shown in any one of SEQ ID NOs: 1-5 (for example, the amino acid sequence shown in any one of SEQ ID NOs: 6-10), or (b) an amino acid sequence in which an inactivating mutation is introduced in the amino acid sequence shown in any one of SEQ ID NOS: 1-5 (for example, the amino acid sequence shown in any one of SEQ ID NOS: 6-10) and 70% or more ( preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more);
is. Mutations include, for example, substitutions, deletions, additions, insertions, etc., preferably substitutions, and more preferably conservative substitutions. The number of mutated amino acids is preferably 1 or more, more preferably 1-8, still more preferably 1-4, and even more preferably 1.

The DNA-binding domain is not particularly limited as long as it contains the N-terminal DNA-binding domain and the C-terminal DNA-binding domain possessed by the inactive Chawanmushroom-derived meganuclease (=monomeric meganuclease). The two domains can be readily determined based on known structural information for meganucleases. The DNA-binding domain may be a homozygous DNA-binding domain (i.e., when the N-terminal DNA-binding domain and the C-terminal DNA-binding domain are from the same species of inactive Pseudomonas meganuclease), or chimeric. It may be a DNA-binding domain (that is, when the N-terminal DNA-binding domain and the C-terminal DNA-binding domain are heterologous to each other from inactive Chahuangtan meganucleases). In a preferred embodiment of the present invention, the DNA-binding domain is at least one DNA-binding domain selected from the group consisting of I-OnuI, I-AaBMI, I-PanMI, I-CpaMI, and I-LtrWI, and In a preferred embodiment, the DNA-binding domain is at least one DNA-binding domain selected from the group consisting of I-OnuI, I-AaBMI, and I-PanMI. In a more preferred embodiment, the DNA-binding domain comprises DNA binding domain of I-OnuI.

The polypeptide of the present invention preferably further comprises a functional domain in addition to the DNA-binding domain of the inactive Chahuangtan meganuclease. When containing a functional domain, the nucleic acid binding factor of the present invention can exhibit activity derived from the functional domain. The functional domain is not particularly limited as long as it is a domain with an amino acid sequence capable of exhibiting a specific function. Deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, sumoylation activity, desumoylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, DNA repair activity, DNA editing activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, nuclease activity, Examples include domains having at least one activity selected from the group consisting of transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, and glycosylase activity. When the polypeptide of the present invention contains functional domains, the number may be one or two or more. Also, functional domains can be employed singly or in combination of two or more.

The polypeptide of the present invention may contain other sequences as long as the nucleic acid binding ability is not significantly impaired (if it also contains a functional domain, as long as the activity of the functional domain is not significantly impaired). Other sequences include, for example, various signal sequences (e.g., nuclear localization signal, nuclear export signal, etc.), protein tags (e.g., biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag, etc.), labeled protein sequences (eg, fluorescent proteins, luminescent enzyme proteins, etc.), protein destabilizing sequences (eg, PEST sequences, etc.), protein stabilizing sequences, transmembrane domains, and the like.

The polypeptide of the present invention may be chemically modified as long as the nucleic acid binding ability is not significantly impaired (if it also contains a functional domain, as long as the activity of the functional domain is not significantly impaired). .

The polypeptide of the present invention may have a carboxyl group (--COOH), carboxylate ( ^--COO- ), amide (--CONH ₂ ) or ester (--COOR) at the C-terminus.

Here, R in the ester includes, for example, C _1-6 alkyl groups such as methyl, ethyl, n-propyl, isopropyl and n-butyl; C _3-8 cycloalkyl groups such as cyclopentyl and cyclohexyl; C _6-12 aryl groups such as , α-naphthyl; phenyl-C _1-2 alkyl groups such as benzyl, phenethyl; C _7- such as α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl ₁₄ Aralkyl group; pivaloyloxymethyl group and the like are used.

In the polypeptide of the present invention, carboxyl groups (or carboxylates) other than the C-terminus may be amidated or esterified. As the ester in this case, for example, the above-described C-terminal ester or the like is used.

Furthermore, in the polypeptide of the present invention, the amino group of the amino acid residue at the N-terminus is protected with a protecting group (for example, a formyl group, a C _1-6 acyl group such as a C _1-6 alkanoyl group such as an acetyl group, etc.). pyroglutamic oxidation of the N-terminal glutamine residue that can be cleaved in vivo, substituents on the side chains of amino acids in the molecule (e.g. -OH, -SH, amino group, imidazole group , indole group, guanidino group, etc.) is protected with an appropriate protecting group (e.g., _C1-6 acyl group such as formyl group, _C1-6 alkanoyl group such as acetyl group, etc.), or the sugar chain is Composite proteins such as bound so-called glycoproteins and the like are also included.

The polypeptides of the invention may be in the form of pharmaceutically acceptable salts with acids or bases. Salts are not particularly limited as long as they are pharmaceutically acceptable salts, and both acid salts and basic salts can be employed. Examples of acid salts include mineral salts such as hydrochloride, hydrobromide, sulfate, nitrate, phosphate; acetate, propionate, tartrate, fumarate, maleate, apple organic acid salts such as acid salts, citrates, methanesulfonates, and paratoluenesulfonates; and amino acid salts such as aspartates and glutamates. Examples of basic salts include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as calcium salts and magnesium salts;

The polypeptides of the invention may be in the form of solvates. The solvent is not particularly limited as long as it is pharmaceutically acceptable, and examples thereof include water, ethanol, glycerol, acetic acid and the like.

The polypeptide of the present invention can be easily produced according to known genetic engineering techniques. For example, it can be produced using PCR, restriction enzyme cleavage, DNA ligation technology, in vitro transcription/translation technology, recombinant protein production technology, and the like.

The nucleic acid-binding factor of the present invention may consist solely of the polypeptide of the present invention, or may contain other substances in addition to the polypeptide of the present invention. Other substances include, for example, functional substances. The functional substance is not particularly limited, and examples thereof include labeling substances, carriers and the like. Functional substances are usually linked directly or indirectly (eg, via a linker) to the polypeptides of the present invention.

Examples of labeling substances include fluorescent substances (e.g., fluorescein, rhodamine, Texas red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM (trademark), Cy (trademark) 3, Cy (trademark) 5, Alexa Fluor (registered trademark) series, etc.), enzymes (eg, β-galactosidase, alkaline phosphatase, glucose oxidase, peroxidase, polyphenol oxidase, etc.).

The carrier is not particularly limited as long as it can carry the polypeptide of the present invention. The material of the carrier is not particularly limited, and examples thereof include organic materials such as various resins and inorganic materials such as silicon materials and metals.

Other substances may be one or a combination of two or more.

The nucleic acid binding agents of the present invention are used to bind nucleic acids containing the recognition sequence of the DNA-binding domain of an inactive Chahuangtan meganuclease.

Recognition sequences differ depending on the type of inactive Chahuanthus meganuclease. For example, a recognition sequence for inactive I-OnuI includes SEQ ID NO: 13, a recognition sequence for inactive I-AaBMI includes SEQ ID NO: 14, and a recognition sequence for inactive I-PanMI includes SEQ ID NO: 15. Recognition sequences for inactive I-CpaMI include SEQ ID NO: 16, and recognition sequences for inactive I-LtrWI include SEQ ID NO: 17. Since the recognition sequence of the inactive Pseudophylum-derived meganuclease is relatively long, the nucleic acid binding factor of the present invention has excellent binding specificity.

Since the DNA-cleaving activity of meganuclease is inactivated, the nucleic acid-binding factor of the present invention binds to nucleic acid but does not cleave the nucleic acid. Therefore, nucleic acid binding allows expression of the activity of the functional domain at the binding site, or utilization of the function of the functional substance.

The field of application of the nucleic acid-binding factor of the present invention is not particularly limited, and includes, for example, fields such as cell therapy, regenerative medicine, and experimental reagents.

In one aspect of the invention, the nucleic acid binding agents of the invention can be used to bind exogenous nucleic acids. The exogenous nucleic acid preferably contains transcription control sequences. That is, the nucleic acid binding factors of the present invention can be used as transcription factors. In this case, for example, it can be used to bind to a specific DNA sequence that does not exist on the human or mouse genome and specifically control the expression of a foreign gene. As another example, a meganuclease target sequence can be introduced in the vicinity of a gene of interest and used to control the expression of that gene. As another example, a reporter gene can be placed downstream of a meganuclease target sequence and used to control the expression of that reporter gene by an artificial transcription factor using an inactivated meganuclease. As yet another example, an inactivated meganuclease can be incorporated into an artificial signal protein and used to translocate into the nucleus and express a gene of interest upon receiving any stimulus.

In one aspect of the present invention, in particular, the intracellular transcription factor domain is liberated by a form (e.g., signal (contact with a specific cell such as a cancer cell) placed in the intracellular domain of a transmembrane protein (eg, a specific gene). can be used as a form designed to control the expression of This form can be used, for example, for cell therapy and tumor immunity (CAR-T therapy, etc.).

In one aspect of the present invention, when the nucleic acid-binding agent of the present invention comprises a carrier, the nucleic acid-binding agent of the present invention is used to purify a nucleic acid containing the recognition sequence of the DNA-binding domain of an inactive Chahuangtan-derived meganuclease. can do. For example, a reagent (nucleic acid binding factor of the present invention) is prepared by binding the polypeptide of the present invention to a resin or magnetic beads, and this is used to purify plasmids, genome fragments, and PCR products into which a meganuclease recognition sequence has been introduced. It can be used as a reagent that can Using this technique, it is possible to develop reagents for preparing libraries for next-generation sequencing.

In one aspect of the present invention, when the nucleic acid-binding factor of the present invention contains a labeling substance, the nucleic acid-binding factor of the present invention is a nucleic acid containing a recognition sequence for a DNA-binding domain of an inactive Chawanthalpus-derived meganuclease (or nucleic acid sequence) can be used for detection. For example, by adding the nucleic acid-binding factor of the present invention to fixed cells or tissue specimens, DNA knocked into the genome can be visualized, and the presence or absence and number thereof can be examined.

(3). Polynucleotides In one aspect, the present invention relates to polynucleotides (also referred to herein as “polynucleotides of the present invention”) that encode the polypeptides of the present invention. This will be explained below.

The coding sequence of the polypeptide of the present invention is not particularly limited as long as it is a polynucleotide consisting of a base sequence encoding the polypeptide of the present invention.

A polynucleotide of the invention, in one aspect thereof, comprises an expression cassette for the polypeptide of the invention.

The expression cassette of the polypeptide of the present invention is not particularly limited as long as it is DNA capable of expressing the polypeptide of the present invention in target cells. A typical example of an expression cassette for a polypeptide of the invention includes a DNA comprising a promoter and a coding sequence for a polypeptide of the invention placed under the control of the promoter.

The biological species from which the target cells are derived is not particularly limited, and examples thereof include bacteria such as Enterobacteriaceae bacteria, fungi such as yeast, animals, and plants. Animals include various mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, pigs, horses, cows, sheep, goats, and deer, as well as non-mammalian vertebrates and invertebrates. mentioned. In a preferred embodiment of the present invention, the biological species from which the target cells are derived include mammals, Escherichia coli, yeast, Bacillus subtilis, zebrafish, medaka, insects, and the like. Also, the type of cells is not particularly limited, and cells derived from various tissues or having various properties, such as blood cells, hematopoietic stem cells/progenitor cells, gametes (sperm, ovum), fibroblasts, epithelial cells, and vascular endothelial cells. , nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells and the like.

The promoter contained in the expression cassette of the polypeptide of the present invention is not particularly limited and can be appropriately selected depending on the target cell. As the promoter, for example, various pol II promoters can be used. Examples of pol II promoters include, but are not limited to, CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, hTERT promoter, β actin promoter, CAG promoter and the like. Other promoters include tryptophan promoters such as trc and tac, lac promoter, T7 promoter, T5 promoter, T3 promoter, SP6 promoter, arabinose-inducible promoter, cold shock promoter, tetracycline-inducible promoter, and the like.

The expression cassette of the polypeptide of the present invention may contain other elements (e.g., multiple cloning site (MCS), drug resistance gene, origin of replication, enhancer sequence, repressor sequence, insulator sequence, reporter protein (e.g., , fluorescent protein, etc.) coding sequence, drug resistance gene coding sequence, etc.). MCS is not particularly limited as long as it contains multiple (eg, 2 to 50, preferably 2 to 20, more preferably 2 to 10) restriction enzyme sites. If the polypeptide of the invention does not contain a functional domain, the MCS can use the coding sequence for any functional domain for insertion.

Drug resistance genes include, for example, chloramphenicol resistance gene, tetracycline resistance gene, neomycin resistance gene, erythromycin resistance gene, spectinomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, puromycin resistance gene and the like.

The reporter protein is not particularly limited as long as it is a luminescent (coloring) protein that reacts with a specific substrate to emit light (coloring) or a fluorescent protein that emits fluorescence upon excitation light. Luminescent (chromogenic) proteins include, for example, luciferase, β-galactosidase, chloramphenicol acetyltransferase, and β-glucuronidase, and fluorescent proteins include, for example, GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred, KillerRed, Cherry, HcRed, mPlum and the like.

The expression cassette for the polypeptide of the present invention may constitute a vector by itself or together with other sequences. The type of vector is not particularly limited, and includes, for example, plasmid vectors such as animal cell expression plasmids; viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, and Sendai viruses; and Agrobacterium vectors. be done. In addition, other vectors include, for example, ColE1-based plasmids typified by pBR322 derivatives in Escherichia coli, pACYC-based plasmids having a p15A origin, pSC-based plasmids, and F-factor-derived mini-F plasmids such as Bac-based plasmids. .

The polynucleotide of the present invention can be easily produced according to known genetic engineering techniques. For example, it can be produced using PCR, restriction enzyme cleavage, DNA ligation technology, and the like.

(Four). Cells In one aspect, the present invention relates to cells containing the polynucleotide of the present invention (also referred to herein as "cells of the present invention").

The cell is the same as the target cell described in "(3) Polynucleotide". Polynucleotides of the invention can be either within the genome of the cell of the invention or extragenomic.

(Five). Reagent In one aspect of the present invention, a reagent (herein referred to as " Also referred to as "the reagent of the present invention").

The reagent of the present invention is not particularly limited as long as it contains at least one selected from the group consisting of the nucleic acid binding factor of the present invention, the polynucleotide of the present invention, and the cell of the present invention. may contain components of Other ingredients are not particularly limited as long as they are pharmaceutically acceptable ingredients. Examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, and binders. , disintegrants, lubricants, thickeners, humectants, coloring agents, fragrances, chelating agents and the like.

The reagent of the present invention may be in the form of a kit. In this case, other reagents, instruments, and the like necessary for using the reagent of the present invention, such as nucleic acid introduction reagents, protein introduction reagents, and buffer solutions, may be included as necessary.

The reagents of the invention can be used, inter alia, to bind nucleic acid binding agents of the invention to exogenous nucleic acids (preferably containing transcription control sequences). In addition, the reagent of the present invention can be used for various applications of the nucleic acid binding factor of the present invention explained in "(2) Nucleic acid binding factor".

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

1. Materials and Methods Unless otherwise specified, the following materials and methods were used in the test examples described later.

1-1. Amino acid sequence, base sequence
1-1-1. Meganuclease sequence from wild-type Pyrophylum
Wild-type I-OnuI amino acid sequence
SRRESINPWILTGFADAEGSFLLRIRNNNKSSVGYSTELGFQITLHNKDKSILENIQSTWKVGVIANSGDNAVSLKVTRFEDLKVIIDHFEKYPLITQKLGDYMLFKQAFCVMENKEHLKINGIKELVRIKAKLNWGLTDELKKAFPEIISKERSLINKNIPNFKWLAGFTSGEGCFFVNLIKSKSKLGVQVQLVFSITQHIKDKNLMNSLITYLGCGYIKEKNKSEFSWLDFVVTKFSDINDKIIPVFQENTLIGVKLEDFEDWCKVAKLIEEKKHLTESGLDEIKKIKLNMNKGRVF（配列番号1）。

Wild-type I-AabMI amino acid sequence
STSDNNGNIKINPWFLTGFIDGEGCFRISVTKINRAIDWRVQLFFQINLHEKDRALLESIKDYLGVGKIHISGKNLVQYRIQTFDELTILIKHLKEYPLVSKKRADFELFNTAHKLIKNNEHLNKEGINKLVSLKASLNLGLSESLKLAFPNVISATRLTDFTVNIPDPHWLSGFASAEGCFMVGIAKSSASSTGYQVYLTFILTQHVRDENLMKCLVDYFNWGRLARKRNVYEYQVSKFSDVEKLLSFFDKYPILGEKAKDLQDFCSVSDLMKSKTHLTEEGVAKIRKIKEGMNRGR（配列番号2）。

Wild-type I-PanMI amino acid sequence
STLESKLNPSYISGFVDGEGSFMLTIIKDNKYKLGWRVVCRFVISLHKKDLSLLNKIKEFFDVGNVFLMTKDSAQYRVESLKGLDLIINHFDKYPLITKKQADYKLFKMAHNLIKNKSHLTKEGLLELVAIKAVINNGLNNDLSIAFPGINTILRPDTSLPQILNPFWLSGFVDAEGCFSVVVFKSKTSKLGEAVKLSFILTQSNRDEYLIKSLIEYLGCGNTSLDPRGTIDFKVTNFSSIKDIIVPFFIKYPLKGNKNLDFTDFCEVVRLMENKSHLTKEGLDQIKKIRNRMNTNRK（配列番号3）。

Wild-type I-CpaMI amino acid sequence
NTSSSFNPWFLTGFSDAECSFSILIQANSKYSTGWRIKPVFAIGLHKKDNELLKRIQSYLGVGKIHIHGKDSIQFRIDSPKELEVIINHFENYPLVTAKQADYTLFKKALDVIKNKEHLSQKGLLKLVGIKASLNLGLNGSLKEAFPNWEELQIDRPSYVNKGIPDPNWISGFASGDSSFNVKISNSPTSLLNKRVQLRFGIGLNIREKALIQYLVAYFDLSDNLKNIYFDLNSARFEVVKFSDITDKIIPFFDKYSIQGKKSQDYQNFKEVADIIKSKNHLTSEGFQEILDIKASMNK（配列番号4）。

Wild-type I-LtrWI amino acid sequence
MINLKNNIEYLNWYICGLVDAEGSFGVNVVKHATNKTGYAVLTYFELAMNSKDKQLLELIKKTFDLECNIYHNPSDDTLKFKVSNIEQIVNKIIPFFEKYTLFSQKRGDFILFCKVVELIKNKEHLTLNGLMKILSIKAAMNLGLSENLKKEFPGCLSVKRPEFGLSNLNKRWLAGFIEGEACFFVSIYNSPKSKLGKAVQLVFKITQHIRDKILIESIVELLNCGRVEVRKSNEACDFTVTSIKEIENYIIPFFNEYPLIGQKLKNYEDFKLIFDMMKTKDHLTEEGLSKIIEIKNKMNTNRI（配列番号5）。

1-1-2. Inactive Pseudomonas meganuclease sequence
Inactive I-OnuI (E22A) (E178A) amino acid sequence
SRRESINPWILTGFADAAGSFLLRIRNNNKSSVGYSTELGFQITLHNKDKSILENIQSTWKVGVIANSGDNAVSLKVTRFEDLKVIIDHFEKYPLITQKLGDYMLFKQAFCVMENKEHLKINGIKELVRIKAKLNWGLTDELKKAFPEIISKERSLINKNIPNFKWLAGFTSGAGCFFVNLIKSKSKLGVQVQLVFSITQHIKDKNLMNSLITYLGCGYIKEKNKSEFSWLDFVVTKFSDINDKIIPVFQENTLIGVKLEDFEDWCKVAKLIEEKKHLTESGLDEIKKIKLNMNKGRVF（配列番号6）。

Inactive I-AabMI (E23A) (E179A) amino acid sequence
STSDNNGNIKINPWFLTGFIDGAGCFRISVTKINRAIDWRVQLFFQINLHEKDRALLESIKDYLGVGKIHISGKNLVQYRIQTFDELTILIKHLKEYPLVSKKRADFELFNTAHKLIKNNEHLNKEGINKLVSLKASLNLGLSESLKLAFPNVISATRLTDFTVNIPDPHWLSGFASAAGCFMVGIAKSSASSTGYQVYLTFILTQHVRDENLMKCLVDYFNWGRLARKRNVYEYQVSKFSDVEKLLSFFDKYPILGEKAKDLQDFCSVSDLMKSKTHLTEEGVAKIRKIKEGMNRGR（配列番号7）。

Inactive I-PanMI (E19A) (E176A) amino acid sequence
STLESKLNPSYISGFVDGAGSFMLTIIKDNKYKLGWRVVCRFVISLHKKDLSLLNKIKEFFDVGNVFLMTKDSAQYRVESLKGLDLIINHFDKYPLITKKQADYKLFKMAHNLIKNKSHLTKEGLLELVAIKAVINNGLNNDLSIAFPGINTILRPDTSLPQILNPFWLSGFVDAAGCFSVVVFKSKTSKLGEAVKLSFILTQSNRDEYLIKSLIEYLGCGNTSLDPRGTIDFKVTNFSSIKDIIVPFFIKYPLKGNKNLDFTDFCEVVRLMENKSHLTKEGLDQIKKIRNRMNTNRK（配列番号8）。

Inactive I-CpaMI (E18A) (D177A) amino acid sequence
NTSSSFNPWFLTGFSDAACSFSILIQANSKYSTGWRIKPVFAIGLHKKDNELLKRIQSYLGVGKIHIHGKDSIQFRIDSPKELEVIINHFENYPLVTAKQADYTLFKKALDVIKNKEHLSQKGLLKLVGIKASLNLGLNGSLKEAFPNWEELQIDRPSYVNKGIPDPNWISGFASGASSFNVKISNSPTSLLNKRVQLRFGIGLNIREKALIQYLVAYFDLSDNLKNIYFDLNSARFEVVKFSDITDKIIPFFDKYSIQGKKSQDYQNFKEVADIIKSKNHLTSEGFQEILDIKASMNK（配列番号9）。

Inactive I-LtrWI (E22A) (E181A) amino acid sequence
MINLKNNIEYLNWYICGLVDAAGSFGVNVVKHATNKTGYAVLTYFELAMNSKDKQLLELIKKTFDLECNIYHNPSDDTLKFKVSNIEQIVNKIIPFFEKYTLFSQKRGDFILFCKVVELIKNKEHLTLNGLMKILSIKAAMNLGLSENLKKEFPGCLSVKRPEFGLSNLNKRWLAGFIEGAACFFVSIYNSPKSKLGKAVQLVFKITQHIRDKILIESIVELLNCGRVEVRKSNEACDFTVTSIKEIENYIIPFFNEYPLIGQKLKNYEDFKLIFDMMKTKDHLTEEGLSKIIEIKNKMNTNRI（配列番号10）。

1-1-3. Other inactive meganuclease sequences
Inactive I-SceI (D44N) (D145A)
MKNIKKNQVMNLGPNSKLLKEYKSQLIELNIEQFEAGIGLILGNAYIRSRDEGKTYCMQFEWKNKAYMDHVCLLYDQWVLSPPHKKERVNHLGNLVITWGAQTFKHQAFNKLANLFIVNNKKTIPNNLVENYLTPMSLAYWFMDAGGKWDYNKNSTNKSIVLNTQSFTFEEVEYLVKGLRNKFQLNCYVKINKNKPIIYSMSYLIFYNLIKPYLKISS1 (sequence number MMYLKLPYNT1).

Inactive I-DmoI (D21A) (E117A)
MHNNENVSGISAYLLGLIIGAGGLYKLKYKGNRSEYRVVITQKSENLIKQHIAPLMQFLIDELNVKSKIQIVKGDTRYELRVSSKKLYYYFANMLERIRLFNMREQIAFIKGLYVAAGDKTLKRLRIWNKNKALLEIVSRWLNNLGVRNTIHLDDHRHGVYVLNISLRDRIKFVHTILSSHLNPLPPE (SEQ ID NO: 12).

1-1-4. Recognition sequences of meganucleases
I-OnuI recognition sequence
TTTCCACTTATTCAACCTTTTA (SEQ ID NO: 13).

I-AabMI recognition sequence
AGGTACCCTTTAAACCTACTAA (SEQ ID NO: 14).

I-PanMI recognition sequence
GCTCCTCATAATCCTTATCAAG (SEQ ID NO: 15).

I-CpaMI recognition sequence
TAGCCCACAATATTAAGGCCAT (SEQ ID NO: 16).

I-LtrWI recognition sequence
AGTAGTGAAGTATGTTATTTAA (SEQ ID NO: 17).

I-SceI recognition sequence
TAGGGATAACAGGGTAAT (SEQ ID NO: 18).

I-DmoI recognition sequence
GCCTTGCCGGGTAAGTTCCGGCGCG (SEQ ID NO: 19).

1-1-5. Other arrays
Notch core sequence
GYLPCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILDYSFTGGAGRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAECEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGVLLSRKRRRQ（配列番号20）。

scFv derived from anti-CD19 antibody
DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKSLQTDDTAIYYCAKHYAMSV2 (seq number GTDTAIYYCAKHYAMSV2).

tTA
MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESG (SEQ ID NO: 22).

1-2. Preparation of artificial transcription factor expression plasmids In order to evaluate the ability of artificial transcription factors to regulate gene expression using inactivated meganucleases, the following artificial transcription factors were prepared. Examples of artificial transcription factors are shown in FIG. 1A. It consists of HA tag sequence, nuclear localization signal (NLS), inactive meganuclease, transcription control domain and proteolytic sequence from the N-terminus. Here, we created an artificial transcription factor using a transcription activation domain (VP48) as the transcription control domain, and evaluated its transcription induction activity in the test examples described later.

The artificial transcription factor consists of a start codon, methionine, a human influenza virus hemagglutinin-derived tag sequence (YPYDVPDYA (SEQ ID NO: 23)), an SV40 large T antigen-derived nuclear localization signal (PKKKRKV (SEQ ID NO: 24)), and a synthetic linker sequence. 1 (GGGSGGS (SEQ ID NO: 25)), an inactive meganuclease sequence (any of SEQ ID NOS: 6-12), a synthetic linker sequence 2 (SGGGGSGGGSGGGGSGGGGS (SEQ ID NO: 26)), a 3x herpesvirus-derived transcription induction domain (PADALDDFDLDMx3 (sequence No. 27)), a mouse ornithine decarboxylase-derived protein destabilizing sequence (HGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 28)) was synthesized as a protein gene linked by insertion of a restriction site. The artificial transcription factor was introduced downstream of the CMV promoter of the gene expression vector pcDNA3 for mammalian cells with a Kozak sequence added to the N-terminus.

1-3. Construction of chimeric Notch receptor containing artificial transcription factor using inactivated meganuclease and expression plasmid of its gene Gene-derived myc tag sequence (EQKLISEEDL (SEQ ID NO: 30)), anti-CD19 antibody-derived scFv (SEQ ID NO: 21), Notch core sequence (SEQ ID NO: 20), inactive I-OnuI sequence (SEQ ID NO: 6), 3x herpesvirus-derived A transcription induction domain (PADALDDFDLDMx3 (SEQ ID NO: 27)), a mouse ornithine decarboxylase-derived protein destabilizing sequence (HGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 28)) was synthesized as a protein gene joined by insertion of restriction sites. The chimeric Notch receptor was introduced downstream of the CMV promoter of the gene expression vector pcDNA3 for mammalian cells with a Kozak sequence added to the N-terminus. For comparison, we also constructed a chimeric Notch receptor in which the inactivated I-OnuI sequence was replaced with tTA (an artificial transcription factor (SEQ ID NO: 22) in which a transcription-inducing domain was added to TetR).

1-4. Construction of Reporter Plasmids In order to evaluate the transcription-inducing activity of artificial transcription factors using inactivated meganucleases and chimeric Notch receptors incorporating them, the following reporter plasmids were constructed. An example of a reporter is shown in FIG. 1B. By placing a CMV mini-promoter downstream of a sequence containing one and seven meganuclease target sequences and placing a reporter gene downstream thereof, binding of an artificial transcription factor using an inactivated meganuclease depends on the binding of the reporter gene. is expressed. Here, luciferase was used as the reporter gene.

Artificial enhancer region containing 1 and 7 meganuclease recognition sequences (any of SEQ ID NOs: 13 to 19), CMV mini-promoter (GGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCC (SEQ ID NO: 31)), Kozak sequence, Emerald luciferase (Eluc) derived from click beetle, It was constructed as a plasmid vector containing, in order, a mouse ornithine decarboxylase-derived protein destabilization sequence (HGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV (SEQ ID NO: 28)) and an SV40 polyadenylation signal. For comparison, a reporter plasmid was also prepared in which the meganuclease recognition sequence was replaced with a tet operator sequence (TCCCTATCAGTGATAGAGA (SEQ ID NO: 32)).

1-5. HEK293T cells, a human embryonic kidney-derived cell line, BHK cells, a hamster kidney-derived cell line, and Raji cells, a human lymphoma, were used in cell culture experiments. HEK293T cells and BHK cells were cultured using DMEM (high glucose) supplemented with 5% calf serum, and Raji cells were cultured using RPMI1640 medium supplemented with 10% calf serum. Also, the cells were cultured in a CO2 incubator under the condition of a _CO2 concentration of 5%.

1-6. Gene transfer into HEK293T and BHK cells
A transfection reagent (Lipofectamine 2000) was used for gene transfer into HEK293 cells and BHK cells. HEK293T cells and BHK cells were plated on Nunc F96 MicroWell White Polystyrene Plates at 1x10^4 cells/100 µL/well, and the next day, 13 ng of reporter plasmid, 13 ng of artificial transcription factor expression plasmid, and 43 ng of artificial transcription factor expression plasmid were transfected using transfection reagents. chimeric Notch receptor expression plasmids alone or in combination were added to each well.

1-7. A simple test of the ability of chimeric Notch receptors to recognize lymphoma To examine the ability of chimeric Notch receptors to recognize lymphomas and express reporter genes, the chimeric Notch receptors and reporter system must be knocked into cells. However, in order to easily evaluate the function of the chimeric Notch receptor, we used a method in which the chimeric Notch receptor and the reporter system were transiently expressed in BHK cells, and the cells were contacted with Raji cells. Specifically, the day after the introduction of the reporter plasmid and the chimeric Notch receptor expression plasmid into the BHK cells, Raji cells were added at 1 x 10^4 cells/100 µL/well.

1-8. Measurement of Reporter Activity A luciferin solution (final concentration 100 μM) and HEPES buffer were added to the medium of transfected HEK293T cells and BHK cells. After that, luciferase-induced luminescence was measured using a microplate reader (SH9000Lab, manufactured by CORONA).

2. Test example
2-1. Induction of reporter gene transcription by artificial transcription factors using inactivated meganucleases
In HEK293T cells, reporter plasmids with 7 recognition sequences for meganucleases I-AaBMI, I-CpaMI, I-LtrWI, I-OnuI, and I-SceI were expressed alone. The reporter transcription activity was examined when the expression plasmid of the artificial transcription factor composed of the activated form was co-expressed (Fig. 2). As a result, in the I-SceI system, yeast-derived I-SceI, which has been studied in particular among meganucleases and is sold as a reagent, is used as an artificial transcription factor and the I-SceI recognition sequence is used as a reporter. A 140-fold increase in transcription-inducing activity was obtained. On the other hand, the I-AaBMI system, which uses the I-AaBMI meganuclease derived from the subphylum Ceratophyta as an artificial transcription factor and the recognition sequence of I-AaBMI as a reporter, yields a 1140-fold increase in transcription induction activity. The I-CpaMI system, which uses the nuclease I-CpaMI as an artificial transcription factor and the I-CpaMI recognition sequence as a reporter, has a 256-fold increase. 278-fold in the I-LtrWI system that uses the recognition sequence of I-OnuI as a reporter, and 1410-fold in the I-OnuI system that uses the meganuclease I-OnuI from the subphylum Chawanthallium as an artificial transcription factor and the recognition sequence of I-OnuI as a reporter Transcriptional induction activity was doubled. In addition, the I-DmoI system, which uses the archaeal meganuclease I-DmoI, which has been extensively studied as well as I-SceI, as an artificial transcription factor and the recognition sequence of I-DmoI as a reporter, induces transcription. No activity could be confirmed (Fig. 3). Based on these results, it was considered that the method of applying the meganuclease derived from the subphylum Chawangalus as a nucleic acid-binding factor, especially to an artificial transcription factor, would be particularly useful.

Next, we confirmed whether artificial transcription factors using inactive meganucleases can have transcription-inducing activity even in reporters that have only one recognition sequence. Specifically, in HEK293T cells, reporter plasmids with one recognition sequence for meganucleases I-OnuI, I-PanMI, and I-SceI were expressed alone, and the corresponding inactivated meganucleases The reporter transcription activity was investigated when the expression plasmid of the artificial transcription factor consisting of was co-expressed (Fig. 4). As a result, it was confirmed that the I-OnuI system had an induction activity of 7.1 times and the I-PanMI system had an induction activity of 7.7 times. From these results, it was confirmed that the meganuclease-based artificial transcription factor has transcription-inducing activity even in reporters that have only one recognition sequence, and that the PanMI system has the same level of transcription activity as the I-OnuI system. rice field.

2-2. Evaluation of Lymphoma Recognition by Chimeric Notch Receptors Containing Inactivated Meganucleases as Artificial Transcription Factors
By modifying the Notch receptor, it is possible to create a chimeric Notch receptor that can specifically recognize lymphoma and induce the expression of any gene (Japanese Patent Publication No. 2018-506293). Animal experiments have confirmed that by knocking in chimeric Notch receptors into T cells, it is possible to create chimeric antigen receptor-expressing T cells that can more accurately recognize and attack cancer cells, making lymphoma treatment safer. (PMID:27693353).

This chimeric Notch receptor is a single transmembrane protein consisting mainly of an anti-CD19 antibody-derived scFv, a Notch receptor-derived polypeptide containing a transmembrane domain, and a tetracycline-responsive repressor-derived artificial transcription factor (tTA). When the CD19 antibody-derived scFv binds to CD19, the Notch receptor-derived polypeptide is cleaved, and finally tTA is released from the membrane and translocated to the nucleus (PMID: 26830878). Translocated to the nucleus, tTA binds to the tet operator sequence and induces the expression of downstream genes. Here, we created a chimeric Notch receptor containing an inactive I-OnuI as an artificial transcription factor instead of tTA, and compared the ability of the existing chimeric Notch receptor to induce the expression of a reporter gene when recognizing lymphoma. It was confirmed by the following experiments that the

Specifically, in the TetR system using the artificial transcription factor tTA and the tet operator as reporters, the addition of lymphoma cells (Raji cells) increased the expression level of the reporter by about 1.3 times, When the I-OnuI system was used, the expression level of the reporter increased 2.1-fold (Fig. 5). In addition, in the cells in which the TetR chimeric Notch receptor and the reporter system were knocked in, the addition of lymphoma cells (Raji cells) increased the transcriptional activity of the reporter more than 15-fold, suggesting that the TetR system is more likely to do so. It has been confirmed that it works well in knock-in cells. Therefore, by using the I-OnuI system instead of the TetR system, chimeric Notch receptors with higher activity could be produced, and chimeric antigen receptor T cells with higher activity could be produced.

Claims (15)

A polypeptide comprising a DNA-binding domain of an inactive Chawanthalpus-derived meganuclease in which mutations are introduced in the LAGLIDADG motif, which is the catalytic center in the N-terminal domain and the C-terminal-side domain of the meganuclease derived from the subphylum of Chahuanthus, Nucleic acid binding factor. (A) said polypeptide further comprises a functional domain; and/or (B) apart from said polypeptide further comprises a functional substance,
The nucleic acid binding factor according to claim 1. (A) The nucleic acid binding agent of claim 1 or 2, wherein said polypeptide further comprises a functional domain. 4. Any one of claims 1 to 3, wherein the meganuclease derived from the subphylum Chahuangtake is at least one selected from the group consisting of I-OnuI, I-AaBMI, I-PanMI, I-CpaMI, and I-LtrWI. The nucleic acid binding factor according to . 5. The nucleic acid-binding factor according to any one of claims 1 to 4, wherein the meganuclease derived from the subphylum Chahuangtake is at least one selected from the group consisting of I-OnuI, I-AaBMI, and I-PanMI. The functional domain comprises transcription promoting activity, transcription repressing activity, methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenyl activity, deadenylation activity, SUMOylation activity, deSUMOylation activity, ribosylation activity, deribosylation activity, myristoylation activity, demyristoylation activity, DNA repair activity, DNA editing activity, DNA damage activity, deamination activity activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, nuclease activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, 4. The nucleic acid binding factor according to claim 2 or 3, which is a domain having at least one activity selected from the group consisting of glycosylase activity and glycosylase activity. 3. The nucleic acid binding factor according to claim 2, wherein said functional substance is at least one selected from the group consisting of labeling substances and carriers. The nucleic acid binding factor according to any one of claims 1 to 7, which is a transcription factor. The nucleic acid binding factor according to any one of claims 1 to 8, wherein said DNA binding domain is a homo DNA binding domain or a chimeric DNA binding domain. A polynucleotide encoding a polypeptide comprising the nucleic acid binding factor of any one of claims 1-9. 11. The polynucleotide of claim 10, comprising an expression cassette for said polypeptide. A cell comprising the polynucleotide of claim 10 or 11. A reagent comprising at least one selected from the group consisting of the nucleic acid binding factor according to any one of claims 1 to 9, the polynucleotide according to claim 10 or 11, and the cell according to claim 12. 14. The reagent of claim 13, for binding said nucleic acid binding agent to an exogenous nucleic acid. 15. The reagent of Claim 14, wherein said exogenous nucleic acid comprises a transcription control sequence.

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