JP2003116559A - New maxizyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new maxizyme that has higher specificity and cleavage activity than those in prior arts to the fused gene specifically expressing in malignant tumor cells, for example, acute promyelocytic leukemia, a method of screening such maxizymes and a method of using the maxizyme as an therapeutic agent for malignant tumors. SOLUTION: The attention is paid to acute promyelocytic leukemia as a malignant tumor, the candidate maxizyme for the PML/RARα fused gene that specifically expresses in such leukemic cells, in other words, the sequence around the Mg+ binding pocket is varied so that it may have a similar stability similar to the inactive form and a variety of candidate maxizymes are designed so that the fused position of the PML/RARα may come closely to the branching position of the sensor arm sequence. Further, the cleavage activity is examined in the cell-free system to prepare the maxizyme having the specific cleavage activity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a maxizyme having a specific cleavage activity for a fusion gene specifically expressed in malignant tumor cells, particularly acute promyelocytic leukemia cells, a screening method for such maxizyme, and Regarding the use of maxizyme.

[0002]

2. Description of the Related Art Acute promyelocytic leukemia (APL) is a subtype of acute myelogenous leukemia (AML), which is estimated to occur in about 15% of AML in Japan and about 500 people / year in Japan as a whole. Has been done. APL is M on FAB classification in AML
It has a large number of coarse granules and a strong dysplastic nucleus in the cell morphology, and often has bundled Auer bodies (Faggot), but in some cases, the granules are delicate and mutated. It recognized. Peroxidase, acetate esterase strong positive, CD33 (+), CD13 (+), HLA-D
R (-), which is considered to be a tumor whose differentiation is stopped at the level of promyelocytic cells in the process of differentiation of myeloid cells. Cytogenetically t (15; 17) (q22; q11) is 80
In more than 100% of cases, this translocation was conversely APL
It is not accepted except.

The characteristic t (15; 17) of APL is 15
PML gene on chromosome 17 and RAR on chromosome 17
(Retinoic acid receptor) Translocation between α genes. Translocation cleavage sites are mainly RARα gene intron 2 and PML
Gene intron 3 to 6. In either case,
A PML / RARα chimeric gene was generated on der (15) and the chimeric protein 110 kD (long form) to 9
0 kD (short form) is produced. This chimeric protein has a DNA-binding domain and a dimer-forming region,
Moreover, PML is a protein that has a retinoic acid binding site.
It forms a heterodimer with RXR and possibly blocks a series of nuclear transcription factor functions of PML and RXR, blocks differentiation of osteoprogenitor cells, and promotes only cell proliferation (dominant negative action). Also PML-RARα
May form a homodimer and act as an original transcription factor, and is considered to have a character as a dominant mutation.

Recent studies using PML / RARα gene-producing transgenic mice have revealed that this chimeric protein plays an important role in the induction of leukemia by APL (Proc. Natl. Acad. Sci. USA).
94, 5302-5307, 1997, Blood 89, 376-387, 1997, Pro
c. Natl. Acad. Sci. USA 94, 2551-2556, 1998). As a very effective drug for the above APL patients, all-trans retinoic acid (all-trans RA) having an effect on differentiation induction of leukemia cells is known (Blood 72, 567-572, 1988). However, remissions in patients treated with all-trans RA alone usually last for only a short period. In addition, such patients often suffer from RA resistant disease, resulting in a serious problem of impeding cell differentiation induction treatment (Blood 82, 1941-1953, 1
993). Therefore, a cure for overcoming RA resistance by APL cells has not yet been established.

On the other hand, position-specified hammerhead ribozyme
Targeting tumorigenic PML / RARα mRNA using
Ting is an approach to overcome RA resistance of APL.
Expected as one. Hammerhead ribozyme
Is a specific site NUX (N = Ika
Ribonucleotides, X = A, C or U) PM
L / RARα can be cleaved (Biochemistry 3
4, 3649-3654, 1995), but this specific site is
Existing on the RARα protein side obtained by the cutting point of the offspring
Therefore, the hammerhead reliance on PML / RARα
Bozyme also has APL specificity for wild-type RARα mRNA.
Is known to also cleave specific PML / RARα mRNA
Teker (Cancer Res. 54, 6365-6369, 1994), Hammer
Tumor formation with head ribozyme PML / RARα mR
A hammer head with the ability to specifically cleave only NA
Designing doribozymes is considered difficult
There is. Novel trans-activated as allosteric
A ribozyme, maxizyme, has a specific RNA sequence
It has a sensor arm that recognizes Mg, which is necessary for cutting. ²⁺Conclusion
Mg in joint area²⁺Mg that traps ions²⁺Combined poke
Via the network, away from the specific RNA sequence recognized
Cleaves target RNA at position (Mol. Cell 2, 617-6
27, 1998). As mentioned above, maxizyme theoretically
Any sequence can be cleaved with few side effects
, Because it can specifically destroy only the target gene,
Currently PML / RARα rather than hammerhead ribozyme
 Maxizymes for various sequences such as mRNA are studied.
Researched and developed.

[0006] In designing maxizymes, several parameters, such as the length of the sequence of the cleavage site and the distance from the effector site to the cleavage site, may be mentioned regarding the selection of the NUX site of the nucleotide of the fusion gene. (Biomacromolecules 1, 108-11
7, 2000). The maxizyme against PML / RARα has already been reported (Biomacromolecules 1,
108-117, 2000), whose cleavage site is PM of the chimeric gene.
It is located on the L portion and has a distance of 4 bases from the PML / RARα fusion site to the branch point of the left and right sensor arm sequences in the active form. This indicates that this maxizyme cleaves the PML part in the presence of the PML / RARα effector in the cell-free system (Biomacromolecules 1, 108-117, 2000). As described above, maxizyme can be designed to specifically cleave any sequence, and has the advantage that it can be specifically cleaved without damaging the normal transcription associated therewith. Therefore, many highly specific gene expressions could be controlled by using maxizymes.

BCR / ABL, PML / RARα and A
ML1 / ETO (Biochem. Biophys. Res. Commun. 21
5, 431-437, 1995, Br. J. Cancer 79, 1325-1331, 199.
9, Blood 92, 1758-1767, 1998, Oncogene 17, 1759-17
68, 1998) and other chimeric genes derived from chromosomal translocations are expressed in leukemia cells and are characterized by inducing leukemia. Therefore, it is considered that the maxizyme against the chimeric gene that induces leukemia recognizes leukemia cells as a reliable target of the ribozyme, like the maxizyme against the chimeric gene derived from the chromosomal translocation. Currently, maxizymes are expected to have strong cleavage activity and high selectivity for target genes.
Recent reports of maxizymes against BCR / ABL have reported that maxizymes act both in vitro and in vivo (Nature 460, 473-474,
2000). Therefore, the action of maxizyme is
It can be controlled allosterically by a sensor arm that specifically recognizes NA, which is a powerful weapon that maxizyme targets and destroys an abnormal chimeric gene, and is suitable for gene therapy to treat future APL patients. Suggests that it is useful.

[0008]

[Problems to be Solved by the Invention] The PML / RARα fusion gene expressed in acute promyelocytic leukemia cells contains:
Long form and short form
It is known that there are two isoforms of rt form) and these are due to the difference in the breakpoints in the PML region (Cell 66, 663-674, 1991). Of the two isoforms, the maxizyme for the long-form PML / RARα fusion gene has already been reported (Biomacromolecules 1, 108-117, 2000).
In such maxizyme, the center of its recognition site (branch point of the left and right sensor arm sequences in the active form) is PML / R.
It was 4 bases away from the fusion site of the ARα fusion gene and was not sufficient in specificity / cleavage activity. The subject of the present invention is a fusion gene specifically expressed in malignant tumor cells such as acute promyelocytic leukemia, a maxizyme having higher specificity and cleavage activity than conventional ones, and a screening method for such a maxizyme, It is intended to provide a method for using such maxizyme such as a therapeutic agent for malignant tumor.

[0009]

[Means for Solving the Problems] In order to solve the above problems, the present inventors have focused on acute promyelocytic leukemia as a malignant tumor, and specifically express the PML / RARα fusion gene in such leukemia cells. Various maxizymes against the maxizyme sensor arm
Designed on the basis of heterodimeric RNA motifs whose catalytic activity is activated by interaction with the sequence of interest of the PML / RARα fusion gene recognized by (see FIG. 1), their cell-free system When the cleavage activity was examined, most maxizymes showed PML / even in the absence of effectors.
It was found to cleave RARα RNA nonspecifically. Based on this, we speculate that the specific cleavage activity may depend on the stability in the inactive form, and we changed the sequence around the Mg ²⁺ binding pocket to make it have the same stability as the inactive form. , Designed various candidate maxizymes so that the fusion site of PML / RARα was located near the branch point of the sensor arm sequence and examined the cleavage activity in the cell-free system. As a result, the maxizyme having a specific cleavage activity was found. Further, from the structural analysis of a candidate maxizyme including maxizyme having such a specific cleavage activity, the basic structure required for specific cleavage activity is determined, and the basic structure is mutated by a few bases maxizyme, that is, the fusion gene. A sensor arm that recognizes a fusion portion of mRNA, a Mg ²⁺ binding pocket that forms an Mg ²⁺ binding pocket in an active form, and an mRNA of the fusion gene
It was found that a maxizyme having a mutated sequence such as a cleavage site arm composed of a nucleotide sequence complementary to the sequence containing the cleavage site has a PML / RARalpha fusion gene. The present invention has been completed by confirming that it is useful as a specific therapeutic agent for acute promyeloacute leukemia.

That is, the present invention relates to malignant tumor cells
It is present in the mRNA of the fusion gene that is specifically expressed and
Bases that have no homology to the base sequences in other mRNAs
NUX in the sequence (N = A, C, G or U, X = A, C or
Is a cleavage site arm sequence that recognizes U) as a cleavage site
And Mg in active form²⁺Mg forming a binding pocket
²⁺Sequence around binding pocket and mRNA of the fusion gene
With a sensor arm array that recognizes the fused portion of
It consists of a terror dimer, and the left and right sensor
The branch point of the genome sequence is the fusion part of the mRNA of the fusion gene.
Various candidate maxizymes located near the position,
Characterized by measuring and evaluating its specificity and cleavage activity
Against the malignant tumor cell-specific fusion gene mRNA
Screening of maxizyme with specific cleavage activity
The method (claim 1) or the malignant tumor cell is a leukemia cell.
Malignant tumor cell-specific according to claim 1, characterized in that
Has specific cleavage activity for mRNA of fusion gene
Maxizyme screening method (Claim 2) or white
Characterizing that the blood diseased cells are acute promyelocytic leukemia cells
The malignant tumor cell-specific fusion gene according to claim 2,
Having a specific cleaving activity for mRNA of Escherichia coli
Screening method (Claim 3) and acute promyelocytic cells
Fusion gene that is specifically expressed in sex leukemia cells
A contract characterized by being a PML / RARα fusion gene
MRNA of fusion gene specific to malignant tumor cell according to claim 3
Of maxizyme having specific cleavage activity against
The training method (claim 4) and candidate maxizymes are
The branch point of the left and right sensor arm arrays in the
Located within 2 bases of fusion site of mRNA of combined gene
Malignant according to any one of claims 1 to 4, characterized in that
Specific cut-off for mRNA of tumor cell-specific fusion gene
Screening method for maxizyme having activity
Requirement 5) or the candidate maxizyme has the sequence CUGANGA
(N = A, C, G or U) and Mg containing the sequence GAAA²⁺
A binding pocket perimeter array is provided.
The malignant tumor cell-specific fusion gene according to any one of 1 to 5
Having a specific cleaving activity for mRNA of Escherichia coli
Screening method (Claim 6) and candidate maxiza
Is characterized by having a stable structure in the inactive form.
The malignant tumor cell characteristic according to any one of claims 1 to 6
It has a specific cleavage activity for mRNA of heterologous fusion gene.
Method for screening maxizyme (claim 7)
Or malignant tumor cell-specific according to any one of claims 1 to 7.
Has specific cleavage activity for mRNA of fusion gene
Maki obtained by screening method of maxizyme
Cyzyme (claim 8) or the following (a) or (b) and (c)
Or consisting of a heterodimer with (d)
Maxizyme (a) From the nucleotide sequence shown in SEQ ID NO: 1
RNA (b) having the nucleotide sequence shown in SEQ ID NO: 1
, One or several bases are deleted, substituted or added.
Heterologous with (c) or (d)
RNA (c) having the enzymatic activity of the imer is shown in SEQ ID NO: 2.
RNA (d) consisting of the nucleotide sequence shown in SEQ ID NO: 2
1 or several bases are deleted in the nucleotide sequence
Consisting of a substituted or added base sequence, and (a) or
Is an RNA whose heterodimer with (b) has enzymatic activity
(Claim 9) and the following (a) or (b) and (c) or (d)
The maxizyme (a) sequence number characterized by consisting of
RNA (b) SEQ ID NO: consisting of the nucleotide sequence shown in No. 3
1 or several bases in the base sequence shown in 3
Consists of a deleted, substituted or added nucleotide sequence,
Heterodimer with (c) or (d) has enzymatic activity
RNA (c) RN consisting of the nucleotide sequence shown in SEQ ID NO: 4
A (d) In the base sequence shown in SEQ ID NO: 4,
Or, bases with some bases deleted, substituted or added
And a heterodimer with (a) or (b)
It relates to RNA having enzymatic activity (claim 10).

The present invention also provides a stabilized maxizyme (claim 11) characterized in that polyA and / or a cap structure is added to the maxizyme according to any one of claims 8 to 10, and claims 8 to 10. A vector capable of expressing the maxizyme according to any one of 10 (claim 12).
Or a malignant tumor cell introduced with the maxizyme according to any one of claims 8 to 10, the stabilized maxizyme according to claim 11, or the vector according to claim 12 (claim 13).
Or using the maxizyme according to any one of claims 8 to 10, the stabilized maxizyme according to claim 11, or the vector according to claim 12, specificity of mRNA of a fusion gene expressed in malignant tumor cells Cleavage method (claim 14), introduction of the maxizyme according to any one of claims 8 to 10, the stabilized maxizyme according to claim 11, or the vector according to claim 12 into malignant tumor cells of a malignant tumor patient. A method for treating a characteristic malignant tumor (claim 15), the maxizyme according to any one of claims 8 to 10, the stabilized maxizyme according to claim 11, or the vector according to claim 12 as an active ingredient. And a therapeutic agent for malignant tumor (claim 16).

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION A method for screening a maxizyme having a specific cleavage activity for mRNA of a malignant tumor cell-specific fusion gene of the present invention includes acute promyelocytic leukemia cells, chronic myelogenous leukemia cells, acute Present in mRNA (target mRNA) of fusion gene specifically expressed in leukemia cells such as myeloid leukemia cells and childhood acute lymphocytic leukemia cells, and malignant tumor cells such as malignant lymphoma cells and acute lymphoblastic leukemia And other MR of human
NU in the base sequence having no homology with the base sequence in NA
A cleavage site arm sequence that recognizes X (N = A, C, G or U, X = A, C or U) as a cleavage site, and a sequence around the Mg ²⁺ binding pocket that forms the Mg ²⁺ binding pocket in the active form. And a heterodimer having a sensor arm sequence that recognizes a fusion portion of the target mRNA,
Various candidate maxizymes in which the branch points of the left and right sensor arm sequences in the active form are located near the fusion site of the target mRNA were prepared, and the specificity and cleavage activity of the candidate maxizyme were measured in vitro (cell-free system) or in vivo. It is not particularly limited as long as it is a method for evaluation, and when a maxizyme having a specific cleavage activity obtained by screening is used, the fusion gene specifically expressed in malignant tumor cells is cleaved at the mRNA stage, and The function can be destroyed.

The fusion gene specifically expressed in the above malignant tumor cells is not particularly limited as long as it is specifically expressed in the malignant tumor cells by translocation of the gene, and PML / RARα (pre-acute) Myeloid leukemia), BCR / ABL (chronic myelogenous leukemia), AML1
/ ETO (MTG8) (acute myeloid leukemia), AML1
/ Evi1 (acute myeloid leukemia), CBFβ / MYH1
1 (acute myeloid leukemia), DEK / CAN (acute myeloid leukemia), Fus / ERG (acute myeloid leukemia), PD
GFRβ / TEL (chronic myelomonocytic leukemia), TEL /
AML1 (pediatric acute lymphocytic leukemia), MLL / AF4
(Acute lymphocytic leukemia), MLL / AF9 (acute lymphocytic leukemia, MLL / ENL (acute lymphocytic leukemia), I
GH / BCL2 (malignant lymphoma), IGH / PAX5
(Malignant lymphoma) IgH / C-MYC (malignant lymphoma), IgH / PRAD1 (malignant lymphoma), ALK /
A fusion gene such as NPM (malignant lymphoma) can be specifically exemplified.

The cleavage site arm sequence in the candidate maxizyme consists of a sequence complementary to the nucleotide sequence containing NUX present in the target mRNA, and the nucleotide sequence containing NUX is substantially the same as the nucleotide sequence in other human mRNA. NU in the base sequence of the target mRNA because it has no homology to
It recognizes X as a cleavage site and, in cooperation with the sensor arm sequence, allows the target mRNA to be specifically and selectively cleaved. For example, when the fusion gene is the PML / RARα isoform, it contains three bases of AUC at positions 476 to 478 in the nucleotide sequence of RARα, which is located 71 bases downstream from the PML / RARα fusion site.
Since the nucleotide sequence of is not homologous to any human sequence, it can be suitably selected as the cleavage site.

As shown in FIG. 1 (top), the sequence surrounding the Mg ²⁺ binding pocket in the candidate maxizyme is such that the cleavage site arm sequence and the sensor arm sequence recognizing the fusion part of the target mRNA are the target mRNA. ²⁺ in active form hybridized to
A sequence forming a binding pocket, for example, when the fusion gene is a PML / RARα isoform, the sequence surrounding the Mg ²⁺ binding pocket is the sequence CUGANGA.
(N = A, C, G or U) and the sequence GAAA as a conserved sequence, and a configuration in which the other portion is mutated is preferable in order to efficiently screen a maxizyme having a specific cleavage activity.

The sensor arm sequence in the candidate maxizyme is composed of a sequence complementary to the fusion part of the target mRNA so that the fusion part of the target mRNA can be recognized. It is configured to be located near the fusion site of the target mRNA, preferably within 2 bases from the fusion site. Targets the branch point of the left and right sensor arm sequences in the active form
The Mg ²⁺ binding pocket in the active form formed has a highly cleaving active structure by being located near the fusion site of RNA, particularly within 2 bases from the fusion site, and has a specific cleaving activity of the target mRNA. The possibility that an excellent maxizyme can be produced increases.

Further, as a candidate maxizyme, inactive type
Maxi with stable structure in (inactive form)
It is preferable to use Zym. Inactive maxizyme
Is Mg²⁺A heterodimer with a binding pocket
Unlike the active maxizyme, which is composed,
As shown in (below), only the cleavage site arm sequence
Hybridizes to the target mRNA,²⁺Combined pocket
Consists of an unformed heterodimer
It And, the stable structure in the inactive type means Mg ²⁺
Complementary base pairs formed between sequences corresponding to binding pockets
Is a structure in which the fusion gene is PML / RA.
In case of Rα isoform
Candidate maxizyme having 8 to 9 complementary base pairs
Results in excellent target mRNA specific cleavage activity
The possibility increases.

Screening of maxizyme having a specific cleavage activity for a target mRNA is carried out in vitro and / or in vivo using various concentrations of candidate maxizymes, but due to its convenience, it is carried out in a cell-free system. It is preferable. The specificity of the above candidate maxizyme is
MRNA of non-fusion gene having the above cleavage site NUX
It is evaluated by whether or not the target mRNA is selectively cleaved without cleaving (normal mRNA), and the cleavage activity of the candidate maxizyme can be evaluated by the concentration of the maxizyme used. The maxizyme of the present invention having a specific cleavage activity against the target mRNA obtained by such a screening method is a leukemia such as acute promyelocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, and childhood acute lymphocytic leukemia, It is useful for treating malignant tumors such as malignant lymphoma. The following is an example of a maxizyme having a high cleavage activity that specifically recognizes a PML / RARα fusion gene that is specifically expressed in acute promyelocytic leukemia cells by taking acute promyelocytic leukemia cells as an example of malignant tumor cells. explain.

In the present invention, the maxizyme which recognizes the PML / RARα fusion gene specifically expressed in acute promyelocytic leukemia cells and has a high cleavage activity is, for example, long form PML / R.
In the case of the ARα fusion gene, a maxizyme comprising a heterodimer of the following (a) or (b) and (c) or (d) can be mentioned. (a) is RNA (MzPRT50L) consisting of the base sequence shown in SEQ ID NO: 1, and (b) is a base in which one or several bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 1. Consists of an array,
And the heterodimer with (c) or (d) is enzymatically active (RN
RNA having an A-cleaving activity), (c) is an RNA having the nucleotide sequence shown in SEQ ID NO: 2 (MzPRT50R)
And (d) is 1 in the base sequence shown in SEQ ID NO: 2.
Alternatively, the heterodimer of (a) or (b) described above is an RNA having a nucleotide sequence in which several bases have been deleted, substituted or added, and which has enzymatic activity. Such (a) or
A maxizyme comprising a heterodimer of (b) and (c) or (d) includes MzPRT50 shown in FIG.
Alternatively, MzPRT50 + G shown in FIG. 18 can be specifically mentioned.

On the other hand, a short form P
In the case of the ML / RARα fusion gene, the following (a) or (b)
And a maxizyme comprising a heterodimer of (c) or (d). (a) is RNA (MzPRK55L) consisting of the nucleotide sequence shown in SEQ ID NO: 3,
(b) consists of the base sequence shown in SEQ ID NO: 3 with one or several bases deleted, substituted or added, and the heterodimer with (c) or (d) shows enzymatic activity. (C) is RNA (MzPRK55R) consisting of the nucleotide sequence shown in SEQ ID NO: 4, which is (d)
Is an RNA having a base sequence in which one or several bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 4, and the heterodimer with (a) or (b) above has an enzymatic activity. Is. With such (a) or (b),
As the maxizyme comprising a heterodimer with (c) or (d), MzPRK55 shown in FIG.
Specific examples thereof include MzPRK55 + G.

As the stabilized maxizyme of the present invention, polyA and / or a cap structure is added to the maxizyme of the present invention obtained by the screening method such as MzPRT50 or MzPRK55 described above, which is protected from degradation by ribonuclease and the like. The cap structure is not particularly limited as long as it has a structure with high property, and the cap structure includes 7-methylguanosine 5 ′ which is 5 ′ → 5 ′ linked to the 5′-terminal nucleoside of maxizyme via a phosphate group. -Phosphate [(7MeG) -5'-ppp-terminal nucleoside] is added to the Cap0 structure, the Cap0 structure is further methylated with OH at the 2'position of the first terminal nucleotide, or the Cap1 structure Furthermore, it illustrates the Cap2 structure that methylates the OH at the 2'position of the second nucleotide. As the stabilized maxizyme, a maxizyme having a structure in which both the cap structure and the poly A structure are added to the 3'side can be more preferably mentioned.

The RNA vector of the present invention is not particularly limited as long as it is a vector capable of expressing the maxizyme of the present invention in cells such as malignant tumor cells, and includes plasmids, viruses, retrotransposons and cosmids. Various vectors can be used, including. In addition, depending on the type of the vector, the RNA sequence that constitutes the heterodimer of maxizyme can be incorporated into the vector as it is, or one or more DNA sequences that are reverse transcripts of the RNA sequence. As a plasmid vector for maxizyme expression, pol II
I promoter (eg, tRNA derived from adenovirus
Or VA-1), CMV promoter, SV40 late promoter, or SV40 early promoter, or the like, and adenovirus vector, adeno-associated virus type 1 or adeno-associated virus. Specific examples include a type 2 vector and a retrovirus vector. Known methods for the production of these vectors (for example, Gene Therapy: Application of Molecular Bio
logy, Elsevier Science Publishing Co., Inc., New York, 1991), for example, by transfecting a malignant tumor cell or the like with a recombinant retroviral vector to encode the maxizyme of the present invention. The gene can be introduced into the chromosome of human cells to efficiently express maxizyme.

The present invention also provides a malignant tumor cell into which the maxizyme, the stabilized maxizyme or the maxizyme expression vector of the present invention has been introduced, or the target m that uses the maxizyme, the stabilized maxizyme or the maxizyme expression vector of the present invention.
The present invention relates to a method for specifically cleaving RNA, and a method for treating a malignant tumor, which comprises introducing the maxizyme, the stabilized maxizyme, or the maxizyme expression vector of the present invention into malignant tumor cells of a malignant tumor patient. Examples of the method for introducing the maxizyme, the stabilized maxizyme, or the maxizyme expression vector of the present invention into malignant tumor cells include a transformation method using calcium phosphate precipitation, a microinjection method, an electroporation method, a lipofection method and the like. When the maxizyme, the stabilized maxizyme, or the maxizyme expression vector of the present invention is introduced into a malignant tumor cell, the target mRNA is specifically cleaved, so that the maxizyme of the present invention is useful for gene therapy or adoptive immunotherapy in patients with malignant tumor. Is.

The therapeutic agent for malignant tumor of the present invention comprises the maxizyme, the stabilized maxizyme or the maxizyme expression vector of the present invention as an active ingredient, and if necessary, a pharmaceutically or physiologically acceptable carrier, excipient or There is no particular limitation as long as it contains a diluent, and leukemias such as acute promyelocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, childhood acute lymphocytic leukemia, and malignant lymphomas and other malignant lymphomas. Since the fusion gene involved in tumor development is cleaved, it can be preferably used for a specific therapeutic method for the above malignant tumor. The therapeutic agent for malignant tumor of the present invention can be administered in various forms such as oral, intravenous, intraperitoneal, intranasal, intradermal, subcutaneous and intramuscular. The effective dose to be administered can be appropriately determined in consideration of the type / composition of a therapeutic agent for malignant tumor, administration method, age and weight of patient, etc., and these can be administered once to several times a day. preferable. When administered by injection such as a vein, the therapeutic agent for malignant tumor of the present invention is prepared by dissolving it in water, but it may be dissolved in physiological saline or glucose solution, etc., if necessary. A buffering agent or a preservative may be added. These formulations may also contain other therapeutically valuable ingredients.

[0025]

The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples. Example 1 (Design and production of maxizyme) Recent studies have revealed the mechanism of transcriptional repression by PML / RARα fusion protein due to the molecular etiology of APL and different reactions in APL by all-trans retinoic acid (RA). (Nature 391, 811-81
4, 1998, Nature 391, 815-818, 1998). Furthermore, PM
Mutation of L / RARα is suspected to be one of the mechanisms responsible for the development of RA resistance in APL patients (Blood 92, 374-382, 1998). Therefore, PML /
Targeting RARα may be useful in the treatment of APL patients, including the case of RA resistance. Above P
Two isoforms of ML / RARα have been clarified so far from the difference in the breakpoints in the PML region. Therefore, we designed several maxizymes that are constructed as heterodimers of two RNA molecules (MzL and MzR) for the two isoforms of PML / RARα. Such a maxizyme has the effect that the sequencer of interest shown in FIG. 1 is recognized by the sensor arm (Mol. Cell 2,
617-627, 1998), and designed based on a heterodimeric RNA motif that is activated by interaction with such effectors.

All of the maxizymes designed above
The following structure is mFold version 3.1 (http: //bioinfo.math.rp
i.edu/~mfold/rna/form1.cgi) (J. M
ol. Biol. 288, 911-940, 1999). 476 to 478th AUC in the nucleotide sequence of RARα is PM
It is located 71 bases downstream from the L / RARα fusion point.
As described above, the 16 nucleotide sequence containing one base was selected as a cleavage site because it has no homology to any human sequence (Nature 330, 444-450, 1987), and P and P in FIG. The non-conserved base indicated by Q was replaced with an appropriate base to stabilize the inactive maxizyme. Note that FIG.
The inner boxed nucleotides represent the conserved sequences required for cleavage activity, and Y and Z are meant to be complementary to the target mRNA sequence.

Cleavage activity and specificity of the maxizyme designed above was examined by a cell-free system. Cleavage reaction in the cell-free system was carried out using 10 μl of reaction buffer [50 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 10 mM MgCl ₂ , 5 ng / μl substrate (PML / RARα RNA having an effector site recognizable by the sensor arm, PML / RARα RNA having no effector site recognizable by the sensor arm, or RARα RNA),
And maxizyme (substrate / maxizyme = 1/1, 1 /
Solution containing 2, 1/4, 1/8, or 1/16)] at 37 ° C. Then, the reaction was stopped by adding 8 μl of stop buffer (US Biochemical Co, Cleveland, OH) to obtain 6% polyacrylamide /
Analyzed on 7M urea gel. As a result, most maxizymes have PML / RARα without effectors.
It was confirmed that RNA was also cleaved. This suggests that the specificity of the cleavage activity depends on the stability of the inactive maxizyme.

The active structure of FIG. 1 has the effector "se".
quence of interest ”molecule, MzL when it has no effector“ sequence of interest ”
And MzR are inactive because they are unable to form Mg ²⁺ binding pockets. In addition, the cleavage activity of the maxizyme designed against PML / RARα is
It was found that the distance from the PML / RARα fusion site to the branch point of the sensor arm in the active form decreased. From the above, the arrangement around the Mg ²⁺ binding pocket is changed, the stability is maintained in the inactive type, and the PML
The maxizyme was redesigned so that the / RARα fusion site was located near the branch point of the sensor arm (Table 1 and Figures 2-15).

[0029]

[Table 1]

The 14 designed maxizymes are described in the literature (Biochem. Biophys. Res. Commun. 215, 431-437, 199).
5) Prepared by in vitro transcription as described. The maxizyme or effector template cDNA contains a bacteriophage T7 RNA polymerase promoter region at their 3'ends. Each maxizyme cDNA was mixed with an equal amount of a DNA oligomer complementary to the bacteriophage T7 RNA polymerase promoter, and 2 mM of A each was added.
TP, GTP, CTP, and UTP (Boehringer Mannh
eim, Indianapolis, IN) and T7 RNA polymerase (New England Biolabs, Beverly, MI) 200 μl of a buffer solution was incubated at 37 ° C. for 4 hours. Then, RQ1 RNase-free DNAse (Promega, M
adison, WI) at 37 ° C. for 15 minutes, and then resuspended in DEPC-treated water. Substrate RARα RNA is also 0.5 mM each of ATP and GTP
And UTP, 0.1 mM CTP, 3.7 MBq
Nucleotide positions 464-737 (EMBL / Genbank: X0 in a buffer containing [α- ³² P] CTP.
6538) (Nature 330, 444-450, 1987) and other RARs
It was prepared by in vitro transcription of a TA cloning vector (Invitrogen, Carlsbad, CA) having an α segment cDNA.

Example 2 (Cleavage activity and specificity of each maxizyme) Next, the cleavage activity and specificity of 14 maxizymes newly designed in Example 1 (FIGS. 2 to 15) were examined in Example 1
The cell-free system was examined in the same manner as described. The results are shown in Table 1. From this result, MzPRT50 (Fig. 1
6) and the structure of MzPRK55 (FIG. 17) show cleavage activity,
It was confirmed that both the specificity was high. In these maxizymes, the recognition site (branch point of active sensor arm) is P
Very close to 2 bases from the fusion site of the ML / RARα fusion gene, and the number of complementary base pairs in the inactive form is 8 each.
Since it was (MzPRT50) or 9 (MzPRK55), it was considered to have a high specific cleavage activity. Active Mg in MzPRT50 and MzPRK55
^The basic structure is the RNA sequence around the ²⁺ binding pocket, and MzP
RT50 + R (Fig. 18) and MzPRK55 + R (Fig. 1
It was also found that a maxizyme having a similar structure obtained by adding or changing several nucleotide sequences to such a basic structure such as 9) has specific cleavage activity. One base of the active Mg ²⁺ binding pocket in each maxizyme was deleted by the method described by Sheldon et al (Nucleic Acids Res. 17,
5679-5685, 1989). It was also confirmed that a maxizyme having a shortened sensor arm or a cleavage site arm, or an extended maxizyme also has a specific cleavage activity.

Example 3 (Effect by Effector) Next, as in Example 1, various concentrations of MzPRT50 were used as maxizyme, various substrates [no mRNA recognized by the sensor arm (No), PML / RARα,
Or RARα alone] and examined the effect of the specific cleavage reaction. The results are shown in Fig. 20. The ratio (Sub / Mz ratio) between the substrate and the maxizyme in FIG. 20 indicates the ratio of the maxizyme concentration to the constant concentration of the substrate. As a result, maxizyme MzPRT50
Which specifically cleaves RARα RNA in, but lacks normal RARα alone and effectors
It was found that RARα has no cleavage activity. Similar to MzPRT50, MzPRK55 specifically cleaved RARα RNA only when the PML / RARα effector was present.

[0033]

EFFECTS OF THE INVENTION Since the maxizyme of the present invention can selectively recognize and cleave mRNA of the fusion gene specifically expressed in malignant tumor cells, it has a specificity / cleaving activity higher than that of conventional maxizymes. Are better. Therefore, the maxizyme, the stabilized maxizyme, the maxizyme expression vector, etc. of the present invention are leukemias such as acute promyelocytic leukemia, chronic myelogenous leukemia, acute myelogenous leukemia, childhood acute lymphocytic leukemia, and malignant lymphomas. It is useful as a therapeutic drug for tumors.

[0034]

[Sequence Listing] SEQUENCE LISTING <110> KEIO UNIVERSITY <120> Novel maxizymes <130> 000000199 <140><141><160> 48 <170> PatentIn Ver. 2.1 <210> 1 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT50L <400> 1 cuuguacuga agacgugccu ccccggcgcc 30 <210> 2 <211> 28 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT50R <400> 2 gggucucaau ggcuucgaaa ugcggggu 28 <210> 3 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK55L <400> 3 cuuguacuga agacguuucc ccugggugau 30 <210 > 4 <211> 28 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK55R <400> 4 uggucucaau ggcuucgaaa ugcggggu 28 <210> 5 <211> 28 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: effector <400> 5 nnnnnnnnnn nnnnnnnnnn nnnnnnnn 28 <210> 6 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzL <400> 6 nnnnnncuga ngannnnnnn nnnnnnnnnn 30 <210> 7 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzR <400> 7 nnnnnnnnnn nnnnnnngaa annnnnnnnn 29 <210> 8 <211> 16 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: RNA including cleavage site <400> 8 nnnnnnnnuh nnnnnn 16 <210> 9 <211> 28 <212> RNA <213> Homo sapiens <400> 9 cgccggggag gcagccauug agacccag 28 <210> 10 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT1L <400> 10 cuuguacuga ugagagcugc cuccccggcg 30 <210> 11 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT1R <400> 11 cugggucuca auggaucgaa augcggggu 29 <210> 12 <211> 16 <212> RNA <213> Homo sapiens <400> 12 accccgcauc uacaag 16 <210> 13 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT2L <400> 13 uguccucuga ugagagcugc cuccccggcg 30 <210> 14 <211 > 29 <212> R NA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT2R <400> 14 cugggucuca auggaucgaa acagacaaa 29 <210> 15 <211> 16 <212> RNA <213> Homo sapiens <400> 15 uuugucuguc aggaca 16 <210> 16 <211> 28 <212> RNA <213> Homo sapiens <400> 16 cacccagggg aaagccauug agacccag 28 <210> 17 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK1L <400> 17 cuuguacuga ugagagcuuu ccccugggug 30 <210> 18 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK1R <400> 18 cugggucuca auggaucgaa augcggggu 29 <210> 19 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK2L <400> 19 uguccucuga ugagagcuuu ccccugggug 30 <210> 20 <211> 29 <212> RNA <213>> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK2R <400> 20 cugggucuca auggaucgaa acagacaaa 29 <210> 21 <211> 28 <212> RNA <213> Homo sapiens <400> 21 caucacccag gggaaagcca uugagacc 28 <210> 22 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK1-22L <400> 22 cuuguacuga agagaguccc cugggugaug 30 <210> 23 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK1-22R <400> 23 ggucucaaug gcuuuucgaa augcggggu 29 <210> 24 <211> 28 <212> RNA <213> Homo sapiens <400> 24 gcaucaccca ggggaaagcc auugagac 28 <210> 25 <211> 31 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK1-33L <400> 25 cuuguacuga ugaggagccc cugggugaug c 31 <210> 26 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK1-33R <400> 26 gucucaaugg cuuuaucgaa augcggggu 29 <210> 27 <211> 28 <212> RNA <213> Homo sapiens <400 > 27 aucacccagg ggaaagccau ugagaccc 28 <210> 28 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK1-54L <400> 28 cuuguacuga agacgauucc ccugggugau 30 <210> 29 <211> 28 <212> RNA <213> Ar tificial Sequence <220><223> Description of Artificial Sequence: MzPRK1-54R <400> 29 gggucucaau ggcuucgaaa ugcggggu 28 <210> 30 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK60L <400> 30 cuuguacuga agaaaucccc ugggugaugc 30 <210> 31 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK60R <400> 31 gucucaaugg cuuucuugaa augcggggu 29 <210 > 32 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK61L <400> 32 cuuguacuga agaaagcccc ugggugaugc 30 <210> 33 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK61R <400> 33 gucucaaugg cuuucuugaa augcggggu 29 <210> 34 <211> 28 <212> RNA <213> Homo sapiens <400> 34 ggcgccgggg aggcagccau ugagaccc 28 <210> 35 <211> 28 <212> RNA <213> Homo sapiens <400> 35 ggggaggcag ccauugagac ccagagca 28 <210> 36 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Ar tificial Sequence: MzPRT51L <400> 36 cuuguacuga ugagaauggc ugccucccc 29 <210> 37 <211> 28 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT51R <400> 37 ugcucugggu cucaucgaaa ugcggggu 28 <210> 38 <211> 31 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT52L <400> 38 cuuguacuga ggaagccgcc uccccggcgc c 31 <210> 39 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT52R <400> 39 gggucucaau ggcuuccuga aaugcggggu 30 <210> 40 <211> 28 <212> RNA <213> Homo sapiens <400> 40 uggcgccggg gaggcagcca uugagacc 28 <210> 41 <211> 32 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT53L <400> 41 cuuguacuga ggacagcccc uccccggcgc ca 32 <210> 42 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT53R <400> 42 ggucucaaug gcuguccuga aaugcggggu 30 <210> 43 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT50S.FL <400> 43 nnnnnncuga agacgugccu ccccggcgcc 30 <210> 44 <211> 28 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRT50S.FR <400> 44 gggucucaau ggcuucgaaa nnnnnnnn 28 <210> 45 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK55S.FL <400> 45 nnnnnncuga agacguuucc ccugggugau 30 <210> 46 <211> 28 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK55S.FR <400> 46 gggucucaau ggcuucgaaa nnnnnnnn 28 <210> 47 <211> 30 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK55 + GS.FL <400> 47 nnnnnncuga agacguuucc ccugggugau 30 <210> 48 <211> 29 <212> RNA <213> Artificial Sequence <220><223> Description of Artificial Sequence: MzPRK55 + GS.FR <400> 48 gggucucaau ggcuucggagaa nnnnnnnn
29

[Brief description of drawings]

FIG. 1 is a diagram showing the basic structure of a designed maxizyme.

FIG. 2 is a diagram showing a basic structure of maxizyme MzPRT1.

FIG. 3 is a diagram showing a basic structure of maxizyme MzPRT2.

FIG. 4 is a diagram showing a basic structure of maxizyme MzPRK1.

FIG. 5 is a diagram showing a basic structure of maxizyme MzPRK2.

FIG. 6 is a diagram showing a basic structure of maxizyme MzPRK1-22.

FIG. 7 is a diagram showing a basic structure of maxizyme MzPRK1-33.

FIG. 8 is a diagram showing a basic structure of maxizyme MzPRK1-54.

FIG. 9 is a diagram showing a basic structure of maxizyme MzPRK55.

FIG. 10 is a diagram showing a basic structure of maxizyme MzPRK60.

FIG. 11 is a diagram showing a basic structure of maxizyme MzPRK61.

FIG. 12 is a diagram showing a basic structure of maxizyme MzPRT50.

FIG. 13 is a diagram showing a basic structure of maxizyme MzPRT51.

FIG. 14 is a diagram showing a basic structure of maxizyme MzPRT52.

FIG. 15 is a diagram showing a basic structure of maxizyme MzPRT53.

FIG. 16 is a diagram showing a basic structure of a standard form of maxizyme MzPRT50.

FIG. 17 is a view showing a basic structure of a standard form of maxizyme MzPRK55.

FIG. 18 is a diagram showing a basic structure of a standard form of maxizyme MzPRT50 + G.

FIG. 19 is a diagram showing a basic structure of a standard form of maxizyme MzPRK55 + G.

FIG. 20 is a diagram showing the results of cleavage activity in the presence or absence of effectors when using MzPRT50 of the present invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 35/02 C12N 9/00 4C086 C12N 5/10 C12Q 1/25 4C087 9/00 C12N 15/00 ZNAA C12Q 1/25 5/00 B (72) Inventor Yasuo Ikeda 35 Shinano-cho, Shinjuku-ku, Tokyo, Keio University School of Medicine (72) Inventor Hiromichi Matsushita Memorial Sloan Ketting Center Center, # 5A210 East 68th St. New York NY10021 USA F-term (reference) 4B024 AA01 BA07 CA04 CA05 CA12 DA02 EA03 EA04 FA02 GA11 HA17 4B050 CC01 CC03 DD11 LL01 4B063 QA01 QQ21 QR35 QR55 QS02 QS31 4B065 AA94X AB01 BA02 CA27 CA44 4C084 AA13 ZB26 ZB27 4C086 AA01 AA02 AA03 EA16 MA01 MA04 NA14 ZB26 ZB27 4C087 AA 01 BC83 CA12 ZB26 ZB27

Claims (16)

[Claims] 1. Another human mRNA which is present in the mRNA of the fusion gene specifically expressed in malignant tumor cells
In the nucleotide sequence having no homology with the nucleotide sequence in
A cleavage site arm sequence that recognizes (N = A, C, G or U, X = A, C or U) as a cleavage site, and a sequence surrounding the Mg ²⁺ binding pocket that forms the Mg ²⁺ binding pocket in the active form. , A heterodimer having a sensor arm sequence that recognizes the fusion portion of the mRNA of the fusion gene, and the branch points of the left and right sensor arm sequences in the active form are located near the fusion site of the mRNA of the fusion gene. A method for screening a maxizyme having a specific cleaving activity for mRNA of a malignant tumor cell-specific fusion gene, which comprises producing a candidate maxizyme and measuring / evaluating its specificity and cleaving activity. 2. The method for screening a maxizyme having a specific cleavage activity for mRNA of a fusion gene specific for a malignant tumor cell according to claim 1, wherein the malignant tumor cell is a leukemia cell. 3. A method for screening a maxizyme having a specific cleavage activity for mRNA of a malignant tumor cell-specific fusion gene according to claim 2, wherein the leukemia cell is an acute promyelocytic leukemia cell. . 4. The malignant tumor cell-specific fusion gene mRNA according to claim 3, wherein the fusion gene specifically expressed in acute promyelocytic leukemia cells is a PML / RARα fusion gene. Method for screening maxizyme having specific cleavage activity with specificity. 5. The candidate maxizyme has an active form in which the bifurcation point of the left and right sensor arm sequences is mR of the fusion gene.
The method for screening a maxizyme having specific cleavage activity for mRNA of a malignant tumor cell-specific fusion gene according to any one of claims 1 to 4, which is located within 2 bases from the fusion site of NA. 6. The candidate maxizyme has the sequence CUGANG.
Mg containing A (N = A, C, G or U) and the sequence GAAA
A screening method for a maxizyme having a specific cleavage activity for mRNA of a malignant tumor cell-specific fusion gene according to any one of claims 1 to 5, which has a sequence around a ²⁺ binding pocket. 7. The malignant tumor cell-specific fusion gene mRNA according to claim 1, wherein the candidate maxizyme has a stable structure in an inactive form.
Method for screening maxizyme having specific cleavage activity against 8. A maxizyme obtained by the method for screening a maxizyme having a specific cleavage activity for mRNA of the malignant tumor cell-specific fusion gene according to any one of claims 1 to 7. 9. A maxizyme comprising a heterodimer of the following (a) or (b) and (c) or (d). (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 (b) In the nucleotide sequence shown in SEQ ID NO: 1, consisting of a nucleotide sequence in which one or several bases have been deleted, substituted or added, and (c ) Or (d) the heterodimer has enzymatic activity RNA (c) consisting of the base sequence shown in SEQ ID NO: 2 (d) The base sequence shown in SEQ ID NO: 2 lacks one or several bases RNA consisting of a deleted, substituted or added nucleotide sequence, and the heterodimer with (a) or (b) has enzymatic activity 10. The following (a) or (b) and (c) or (d)
A maxizyme characterized by comprising and. (a) RNA consisting of the nucleotide sequence shown in SEQ ID NO: 3 (b) In the nucleotide sequence shown in SEQ ID NO: 3, consisting of a nucleotide sequence in which one or several bases have been deleted, substituted or added, and (c ) Or (d) a heterodimer having an enzymatic activity RNA (c) consisting of the base sequence shown in SEQ ID NO: 4 (d) The base sequence shown in SEQ ID NO: 4 lacks one or several bases RNA consisting of a deleted, substituted or added nucleotide sequence, and the heterodimer with (a) or (b) has enzymatic activity 11. A stabilized maxizyme which is characterized in that polyA and / or a cap structure is added to the maxizyme according to any one of claims 8 to 10. 12. A vector capable of expressing the maxizyme according to claim 8. 13. A malignant tumor cell introduced with the maxizyme according to any one of claims 8 to 10, the stabilized maxizyme according to claim 11, or the vector according to claim 12. 14. A fusion gene mRNA expressed in a malignant tumor cell, which comprises using the maxizyme according to any one of claims 8 to 10, the stabilized maxizyme according to claim 11, or the vector according to claim 12. Specific cleavage method. 15. A malignant tumor characterized by introducing the maxizyme according to any one of claims 8 to 10, the stabilized maxizyme according to claim 11 or the vector according to claim 12 into malignant tumor cells of a malignant tumor patient. How to treat a tumor. 16. A therapeutic agent for malignant tumor, comprising the maxizyme according to any one of claims 8 to 10, the stabilized maxizyme according to claim 11 or the vector according to claim 12 as an active ingredient.