Enzyme, enzyme complex, and use thereof

Abstract

Provided are: an enzyme having the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell; a complex of the enzyme; and use of the same. In the present invention, an enzyme having the activity of converting guanosine in a polynucleotide into adenosine is used in an animal cell.

Description

Enzymes, enzyme complexes, and their uses

[0001] The present invention relates to an enzyme and an enzyme complex having the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in animal cells, and to the use of these enzymes and enzyme complexes.

[0002] DNA editing is one of the treatment methods for genetic diseases. Although DNA editing is an effective treatment method, there are concerns about the occurrence of unexpected mutations called off-target mutations. Because DNA (genome) is a molecule that persists permanently within cells, if a mutation occurs in DNA, there is a concern that the mutation may be passed down through generations.

[0003] In recent years, RNA editing has attracted attention as a method for resolving such concerns. mRNA is not only a temporary molecule copied from DNA, but also has a mechanism within cells that degrades incorrect mRNA. Therefore, even if a mutation occurs in mRNA due to off-targeting, the mRNA is quickly degraded, eliminating the concern that the mutation will be passed down through generations. Therefore, RNA editing is considered to be a highly safe and effective method for treating genetic diseases and the like.

[0004] RNA editing is a physiological process. In mammals, RNA editing that converts adenosine to guanosine and RNA editing that converts cytidine to uridine are known, while in plants, RNA editing that converts cytidine to uridine and RNA editing that converts uridine to cytidine are known.

[0005] Among these RNA editing processes, the RNA editing that converts adenosine to guanosine and the RNA editing that converts cytidine to uridine are known to be hydrolytic deamination reactions of nucleic acid bases. Furthermore, among the enzymes that catalyze the conversion of cytidine to uridine, there are enzymes that not only have the activity of converting cytidine in RNA to uridine, but also have the activity of converting cytidine in DNA to uridine.

[0006] In RNA editing, adenosine in mRNA is converted to guanosine by adenosine deaminase (ADAR). Since inosine forms a Watson-Crick base pair with cytosine, the inosine is recognized as the same genetic code as guanosine in translation (Non-Patent Document 1).

[0007] Plants have an RNA editing mechanism that converts cytidine to uridine, and it is known that RNA editing is catalyzed by a type of PPR (pentatricopeptide repeat) protein that binds to RNA in a sequence-specific manner. PPR proteins are characterized by repeats of a PPR motif with two α-helical structures consisting of approximately 35 amino acids. Among PPR proteins, PPR-DYW type PPR proteins, which have an E (extension) domain and a DYW (Asp-Tyr-Trp) domain at the carboxyl terminus, have deaminase activity and catalyze RNA editing that converts cytidine to uridine via hydrolytic deamination of cytidine.

[0008] The present inventors have discovered an enzyme from hornwort (Anthoceros agrestis) that catalyzes the reaction of converting uridine to cytidine. Furthermore, the present inventors have discovered that the GRP (Gly-Arg-Pro) domain of this enzyme catalyzes the reaction of converting uridine to cytidine (Patent Document 1).

[0009] Furthermore, the present inventors analyzed the base sequence of the total RNA of Arabidopsis thaliana, a model plant, and found that in addition to the above-mentioned base substitutions, there were many guanosine-to-adenosine substitutions, and that the incidence of these substitutions was significantly higher than the incidence of mutations (Non-Patent Document 2).

[0010] WO2023 / 120658 A1

[0011] Md TA Azad et al., “Site-directed RNA editing by adenosine deaminase acting on RNA for correction of the genetic code in gene therapy” Gene Ther., 24(12) 779-786 (2017)Ruchika et al., “Genome-Wide Identification of U-to-C RNA Editing Events for Nuclear Genes in Arabidopsis thaliana” Cells, 10(3), 635 (2021)

[0012] However, to date, the mechanism for converting guanosine in polynucleotides into genetic code equivalent to adenosine in animal cells and the enzymes that catalyze this conversion have not been identified, and it has not been possible to convert guanosine in polynucleotides into genetic code equivalent to adenosine in animal cells.

[0013] An object of one aspect of the present invention is to provide an enzyme and an enzyme complex having the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, and uses thereof.

[0014] Hornwort is known to contain PPR proteins involved in RNA editing, including DYW-type PPR proteins that catalyze the deamination of cytidine, GRP-type PPR proteins that catalyze the transamination of uridine, and DRH (Asp-Arg-His)-type PPR proteins. However, the function and role of DRH-type PPR proteins remained unknown.

[0015] As a result of extensive research, the inventors discovered that the DRH-type PPR protein has the activity of converting guanosine in a polynucleotide into a genetic code equivalent to adenosine in animal cells (e.g., human cells), and thus completed the present invention.

[0016] The activity of converting guanosine in a polynucleotide into the genetic code equivalent to adenosine is thought to be derived from the deamination of guanosine to produce xanthosine, and more specifically, from the formation of a Watson-Crick base pair with the uridine of the tRNA anticodon. Xanthosine, a deamination product of guanine, is known to be recognized as either guanosine or adenosine by Sanger sequencing analysis, and this is thought to be due to the base pairing of xanthosine with cytosine and thymine (Non-Patent Document 1).

[0017] In order to solve the above problems, one aspect of the present invention includes the following inventions.

[0018] [1] An enzyme having the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell.

[0019] [2] An enzyme complex comprising the enzyme according to [1] and a base sequence recognition module that binds to a specific sequence within a polynucleotide, thereby allowing the enzyme to act on a specific guanosine within the polynucleotide sequence.

[0020] [3] An expression vector for animals, comprising a polynucleotide encoding the enzyme complex according to [2].

[0021] [4] A genetic disease therapeutic agent for animals, comprising the enzyme complex according to [2] or the expression vector according to [3].

[0022] According to one aspect of the present invention, there are provided an enzyme and an enzyme complex having the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, and uses thereof.

[0023] 1 is a diagram showing the structure of an expression vector in an example of the present invention; 2 is a diagram showing the function of the PPR domain of the PPR56 protein in an example of the present invention; 3 is an image showing the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an example of the present invention;

[0024] One embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the respective configurations described below, and various modifications are possible within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference. Furthermore, unless otherwise specified in this specification, "A to B" representing a numerical range means "greater than or equal to A and less than or equal to B."

[0025] [1. Enzymes] An enzyme according to one embodiment of the present invention is an enzyme that has the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell. An enzyme according to one embodiment of the present invention may also be an enzyme that has the activity of deaminating guanosine in a polynucleotide and converting it to xanthosine in an animal cell. Xanthosine in a polynucleotide can be translated as the same genetic code as adenosine during protein biosynthesis. As used herein, the term "polynucleotide" encompasses DNA and RNA.

[0026] Various intracellular mechanisms differ between microorganisms (e.g., Escherichia coli) and animal cells (e.g., human cells), and therefore, metabolism and reactions occurring in microorganisms do not necessarily occur in the same way in animal cells. For example, codon usage, protein stability, the presence or absence of organelles, the presence or absence of splicing, and gene expression mechanisms differ between microorganisms and animal cells, and therefore, metabolism and reactions occurring in microorganisms do not necessarily occur in the same way in animal cells. As demonstrated in the examples described below, an enzyme according to one embodiment of the present invention is expressed in animal cells. Therefore, an enzyme according to one embodiment of the present invention can convert guanosine in a polynucleotide into a genetic code corresponding to adenosine in animal cells and / or can deaminate guanosine in a polynucleotide to convert it to xanthosine in animal cells.

[0027] There are no limitations on the method for confirming whether a desired enzyme (i) has the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, or (ii) has the activity of deaminating guanosine in a polynucleotide and converting it to xanthosine in an animal cell.

[0028] For example, (i) converting the start codon "ATG" of a marker gene (e.g., a gene encoding a fluorescent protein) to "GTG" and then inserting the marker gene into an expression vector to prepare expression vector A; (ii) introducing expression vector A and expression vector B for expressing a desired enzyme into animal cells; (iii) confirming the expression of a marker protein encoded by the marker gene in the animal cells. If expression of the marker protein can be confirmed in (iii) above, this means that the start codon lost by mutation has been restored, i.e., the mutated guanosine has been recognized as adenosine, and it can be determined that the desired enzyme has the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in animal cells, or has the activity of deaminating guanosine in a polynucleotide and converting it to xanthosine in animal cells.

[0029] For example, (iv) an expression vector B for expressing a desired enzyme is introduced into an animal cell, (v) a cDNA corresponding to the RNA of a specific gene is obtained in the animal cell, and (vi) the base sequence of the cDNA is decoded. If, in (vi), the frequency with which a base that should be guanosine is decoded as adenosine increases compared to when expression vector B is not introduced into the animal cell, it can be determined that the desired enzyme has the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in the animal cell, or has the activity of deaminating guanosine in a polynucleotide into xanthosine in the animal cell.

[0030] The present invention has the following advantages (a) to (c), for example: (a) It is possible to provide an enzyme and an enzyme complex having an activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell, and uses thereof.

[0031] (b) A mutated polynucleotide can be repaired to a normal polynucleotide by converting the guanosine that has been generated in the polynucleotide due to the mutation into the genetic code equivalent of adenosine.

[0032] (c) By converting guanosine, which is important for the onset and / or progression of disease, into a genetic code equivalent to adenosine, the onset and / or progression of disease can be suppressed. For example, in animals, including humans, genetic information is separated in DNA by intervening sequences called introns. After transcription, mature mRNA is generated from pre-mRNA by RNA splicing. The sequences at the boundaries between exons and introns that code for protein information are well conserved, often Ag and gG. (Uppercase letters indicate exons, and lowercase letters indicate introns.) If this conserved A mutates to guanosine, the exon is no longer spliced. It is also known that splicing involves the binding of g at the 5' end of an intron to a specific a in the intron; if the a at this branch point mutates to g, splicing also ceases. Many cases of diseases caused by such splicing abnormalities are known, and converting these adenosines to guanosine is expected to improve these diseases. In addition, high expression of TERRA (Telomeric Repeat-Containing RNA), a long non-coding RNA expressed from telomeres at the ends of chromosomes, is known to be involved in the progression of cancer. Furthermore, the function of TERRA is known to be involved in a rigid structure called a guanine quadruplex. In a guanine quadruplex, four guanine molecules form hydrogen bonds with each other, resulting in a rigid planar structure. By converting guanosine in TERRA to a genetic code equivalent to adenosine (deamination of guanosine in TERRA), the hydrogen-bonded guanine quadruplex structure is disrupted, which is expected to result in the inhibition of cancer progression.

[0033] An enzyme according to one embodiment of the present invention may be an enzyme containing any one of the following polypeptides (1) to (6). With this configuration, guanosine in a polynucleotide can be converted into a genetic code corresponding to adenosine.

[0034] (1) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2; (2) A polypeptide consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1 or 2; (3) A polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2; (4) A polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 4; (5) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 4; (6) A polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 4.

[0035] Specific sequences of SEQ ID NOs: 1 to 4 are shown below. Note that a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 is presented as an example of a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, and a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 4 is presented as an example of a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

[0036] SEQ ID NO: 1: KKPAKACIEVKNRVHEFTVGEERSDVASMLRDVSTKLKLGGHVPETWLVLKAASEQEKEDALCGHAEKLALAYGLLNTPDGTTLLVTKNLRMCHDCHSSTKIMSHVENREIIVRDVHRVHRFLNGACSCGDRH; SEQ ID NO: 2: KKAAKACIEVQNIEVHEFTVGEDRSDISSKLRSVNMRLKEEGGHVPQTQLVLKAMSEEKKEDALCGHAEKLALAYGLLNTPDGTTLVVTKNLRMCNDCHSSTKIMSRLEKREIIVRDAHRVHRFLDGACSCRDRH; SEQ ID NO: 3: AAGAAGCCAGCCAAGGCATGCATCGAGGTGAAGAACAGGGTGCATGAGTTCACAGTGGGTGAAGAGAGGTCTGATGTTGCATCCATGCTGAGGGATGTGAGTACGAAGTTGAAATTGGGCGGCCATGTTCCGGAAACATGGCTGGTCCTGAAGGCAGCTCTGAGCAGGAGAAGGAAGACGCTTTGTGTGGACATGC CGAGAAGCTGGCTCTGGCATATGGTCTGCTCAACACTCCAGATGGTACAACTCTGCTAGTGACTAAAAACCTGCGCATGTGCCATGACTGCCACAGCAGCACCAAGATTATGTCACATGTGGAGAATCGAGAAATCATAGTAAGAGATGTACACCGAGTGCATCGTTTTCTGAATGGTGCTTGCTCTTGGGGATCGCCAC;SEQ ID NO: 4: AAGAAGGCAGCCAAGGCATGCATCGAGGTGCAGAACATAGAGGTGCATGAGTTCACAGTGGGTGAAGATAGGTCTGATATTTCATCCAAGCTGAGGAGTGTGAATATGCGGTTGAAAGAGGAGGGCGGCCATGTTCCGCAAACACAGCTGGTCCTGAAGGCAATGTCTGAGGAGAAGAAGGAAGACGCTTTGTGTGGACA TGCCGAGAAGCTGGCTCTGGCATATGGTCTGCTCAACACTCCAGATGGTACAACTCTGGTAGTGACTAAAAACCTGCGCATGTGCAATGACTGCCACAGCAGCACCAAGATTATGTCACGTCTGGAGAAACGAGAAATCATAGTAAGAGATGCACACCGAGTGCATCGTTTTCTGGATGGTGCTTGCTCTTGTAGGGATCGCCAC. ;

[0037] The present inventors searched for PPR-DYW homologs in the hornwort genome and identified a PPR protein having a DRH domain instead of the DYW domain. As shown in the Examples below, the DRH domain linked to the E2 domain (E2-DRH) is an enzyme that catalyzes a reaction in human-derived cells to deaminate any guanosine and convert it to xanthosine, resulting in the same genetic code as adenosine. The amino acid sequence of the E2-DRH domain is represented, for example, by the amino acid sequence of SEQ ID NO: 5 combined with SEQ ID NO: 1, or the amino acid sequence of SEQ ID NO: 5 combined with SEQ ID NO: 2. As long as the enzyme has the activity of converting guanosine to xanthosine, it may be an E2-DRH-like protein of a plant other than hornwort, an enzyme obtained by modifying cytidine deaminase or adenosine deaminase to act on guanosine, or an enzyme obtained by modifying an enzyme that deaminates free guanine or guanosine to act on guanosine in a polynucleotide.

[0038] The enzyme according to one embodiment of the present invention is more preferably an enzyme containing an E2 domain, which is any one of the polypeptides (7) to (12). With this configuration, the activity of the enzyme can be increased, and guanosine in a polynucleotide can be more efficiently converted into the genetic code corresponding to adenosine.

[0039] (7) A polypeptide consisting of the amino acid sequence of SEQ ID NO: 5; (8) A polypeptide consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 5; (9) A polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 5; (10) A polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 6; (11) A polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a base sequence complementary to the polynucleotide consisting of the base sequence of SEQ ID NO: 6; (12) A polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 6.

[0040] Specific sequences of SEQ ID NOs: 5 and 6 are shown below. The polynucleotide consisting of the base sequence of SEQ ID NO: 6 is an example of a gene encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5.

[0041] SEQ ID NO: 5: AATYVLLSNIYAEAGKWDMVSWVRTMMRERGIR; SEQ ID NO: 6: GCTGCTACCTATGTTCTCCTTTCCAACATCTATGCTGAAGCTGGAAAGTGGGACATGGTATCATGGGTGCGGACTATGATGCGCGAGAGAGGGATTCGC.

[0042] The E2 domains shown in SEQ ID NOs: 5 and 6 are derived from DRH-type PPR proteins of Hornwort. However, the E2 domains are not limited to those derived from Hornwort. The E2 domains may also be derived from DYW-type or GRP-type PPR proteins.

[0043] From the viewpoint of further increasing the enzyme activity, the E2 domain is preferably located on the amino-terminal side of the DRH domain. The E2 domain and the DRH domain may be linked directly or via a desired linker (e.g., an amino acid or a polypeptide).

[0044] With regard to (2) and (8) above, "substitution, deletion, insertion, and / or addition of one or several amino acids" refers to the substitution, deletion, insertion, and / or addition of a number of amino acids (preferably 50 or less, more preferably 40 or less, more preferably 30 or less, more preferably 20 or less, more preferably 10 or less, more preferably 9 or less, more preferably 8 or less, more preferably 7 or less, more preferably 6 or less, more preferably 5 or less, more preferably 4 or less, more preferably 3 or less, more preferably 2 or less, and most preferably 1 or less) that allows the original function of the polypeptide to be maintained. The "substitution, deletion, insertion, and / or addition of one or several amino acids" may include artificially induced mutations and naturally occurring mutations.

[0045] With respect to (3), (6), (9), and (12) above, the sequence identity is preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and most preferably 99% or more.

[0046] The determination of sequence identity between two sequences can, for example, be accomplished using a mathematical algorithm. Such mathematical algorithms include, but are not limited to, the algorithm of Myers and Miller (1988) CABIOS 4:11-17; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2:482; the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; and the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, with modifications as described in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

[0047] With respect to (5) and (11) above, "stringent conditions" refers to conditions under which so-called specific hybrids are formed and nonspecific hybrids are not formed. Examples of stringent conditions include conditions under which highly homologous DNAs (e.g., DNAs having a homology of 50% or more, 65% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more) hybridize with each other, but DNAs having lower homology do not hybridize with each other, or conditions under which washing is performed once, preferably two to three times, at a salt concentration and temperature corresponding to the washing conditions of conventional Southern hybridization: 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS.

[0048] [2. Enzyme Complex] An enzyme complex according to one embodiment of the present invention comprises an enzyme according to one embodiment of the present invention and a base sequence recognition module that binds to a specific sequence in a polynucleotide and thereby causes the enzyme to act on a specific guanosine in the polynucleotide sequence. With this configuration, it is possible to convert a desired guanosine in the polynucleotide into a genetic code corresponding to adenosine in an animal cell, and to deaminate the desired guanosine in the polynucleotide and convert it to xanthosine in the animal cell.

[0049] The guanosine on which the enzyme acts is not limited, and may be, for example, a guanosine closely related to the pathology of a disease (e.g., a guanosine in TERRA), or a guanosine present in a codon that has been mutated so as to encode an amino acid different from the wild-type amino acid.

[0050] The base sequence recognition module may be (a) one that binds to a specific sequence in a polynucleotide that is involved in the cause or exacerbation of a pathological condition, and / or (b) one that binds to a specific sequence in a polynucleotide that has a mutation in which adenosine is replaced with guanosine. With this configuration, it is possible to ameliorate the pathological condition and / or suppress the synthesis of mutant proteins and promote the synthesis of normal proteins.

[0051] The base sequence recognition module is not particularly limited as long as it allows the enzyme to act on a guanosine present at a predetermined position in a polynucleotide. The base sequence recognition module may be a single molecule or a complex of multiple molecules. For example, the base sequence recognition module may include either a protein that binds sequence-specifically to a polynucleotide or a polynucleotide that is complementary to at least a portion of the sequence.

[0052] More specifically, when the base sequence recognition module is a polynucleotide, examples of the base sequence recognition module include an MS2 system in which RNA complementary to a portion of a target RNA is bound; a CRISPR-dCas system using dCas9 or 13 or the like in which the nucleic acid cleavage ability has been inactivated; and an RNA or DNA complementary to a portion of a target RNA bound to a polynucleotide sequence that specifically binds to a specific nucleic acid-binding protein.

[0053] When the base sequence recognition module is a protein, examples of the base sequence recognition module include a zinc finger motif, a TAL effector, a PPR protein, and a peptide nucleic acid (PNA) complementary to a target RNA.

[0054] The CRISPR-dCas system uses a dCas protein that has lost its nuclease and nickase activities, and a guide RNA. The guide RNA corresponds to the CRISPR RNA (crRNA) and contains a complementary base sequence that forms base pairs with the target sequence and a base sequence that functions as a transactivating crRNA (tracrRNA) and serves as a scaffold for the binding of the dCas protein. The guide RNA contains the crRNA sequence, which allows it to bind to the dCas protein, forming a dCas-guide RNA complex. The enzyme can be made to act specifically on the target guanosine by using a fusion protein of the enzyme and the dCas protein and a guide RNA designed for a guanosine closely related to the pathology of a disease or a guanosine generated in a polynucleotide by an A to G mutation.

[0055] The zinc finger motif is a combination of multiple Cys2His2-type zinc finger units, each with a different structure, and has the property of binding specifically to a base sequence. By using a fusion protein of the enzyme with a zinc finger motif designed to bind to a guanosine closely related to the pathology of a disease or to the vicinity of a guanosine generated in a polynucleotide by an A to G mutation, the enzyme can be made to act specifically on the target guanosine.

[0056] The TAL effector has a repeating structure of modules each consisting of approximately 34 amino acids, and the binding stability and base specificity are determined by the 12th and 13th amino acid residues of each module. By using a fusion protein of the above enzyme with a TAL effector designed to bind to a guanosine closely related to the pathology of a disease or to the vicinity of a guanosine generated in a polynucleotide by A to G mutation, the above enzyme can be made to act specifically on the target guanosine.

[0057] Like the TAL effector, the PPR protein can be configured to recognize a specific base sequence by a series of PPR motifs that recognize a single nucleic acid base. By using a fusion protein of the enzyme and a PPR protein designed to bind to a guanosine closely related to the pathology of a disease or to the vicinity of a guanosine generated in a polynucleotide by an A to G mutation, the enzyme can be made to act specifically on the target guanosine.

[0058] In the backbone of PNA, N-(2-aminoethyl)glycine, rather than sugar, is bound via an amide bond. In PNA, the purine ring and pyrimidine ring corresponding to the nucleic acid base are bound to the backbone via a methylene group and a carbonyl group, and like polynucleotides, they can bind complementarily to a target polynucleotide in a base sequence-specific manner. By using a fusion protein of the enzyme and a PNA designed to be complementary to guanosine closely related to the pathology of a disease or to the vicinity of a guanosine generated in a polynucleotide by A to G mutation, the enzyme can be made to act specifically on the target guanosine.

[0059] In the enzyme complex, the enzyme and the base sequence recognition module may be linked via a covalent bond or by a compound, a linker, or the like. The linker may be a functional group or molecule that links the enzyme and the base sequence recognition module. For example, the linker may link the enzyme and a protein or nucleic acid that binds sequence-specifically to a polynucleotide. The linker may be disposed between, for example, two types of functional groups, two types of molecules, or two types of other moieties and covalently bonded to each other. The linker may be a peptide chain consisting of one amino acid or multiple amino acids, or may be an oligonucleotide. The linker may be an organic molecule, a functional group, a polymer, or the like.

[0060] Below, we will explain the case where a PPR protein is used as the base sequence recognition module and the case where the MS2 system is used. Note that these base sequence recognition modules are merely examples, and the present invention is not limited to these.

[0061] PPR proteins are RNA-binding proteins widely found in plants and contain repeats of a PPR motif with two α-helical structures consisting of approximately 35 amino acids. PPR proteins can be configured to recognize and bind to specific base sequences through a series of PPR motifs that recognize a single nucleic acid base, making them useful as base sequence recognition modules. By fusing a PPR protein fragment with a PPR motif designed to recognize a base sequence near the target guanosine with the enzyme, the enzyme can be directed to the target guanosine, thereby enabling deamination of the target guanosine. Preferably, the PPR motif recognizes a base sequence immediately preceding the target guanosine. For example, the PPR motif may recognize a sequence of seven or more bases, such as up to one, two, three, four, or five bases upstream of the target guanosine. A fusion protein of a PPR motif that recognizes a target base sequence and an enzyme binds to the vicinity of the target guanosine in a specific polynucleotide via the PPR motif in animal cells (e.g., human cells). For example, when the active site of an enzyme is induced at a guanosine that is closely related to the pathology of a disease or a guanosine that has been generated in a polynucleotide by an A to G mutation, conversion of the target nucleic acid base can be efficiently carried out.

[0062] The genome of MS2 phage, an RNA virus, is a single-stranded RNA that functions as a positive-strand mRNA. When MS2 phage infects Escherichia coli, negative-strand RNA is synthesized following translation of a gene necessary for MS2 phage proliferation, and positive-strand RNA is then synthesized using this negative-strand RNA as a template. MS2 phage synthesizes various proteins based on the positive-strand RNA, and among these proteins, the MS2 coat protein is a protein that constitutes the shell of MS2 phage and has the property of binding specifically and strongly to loop RNA present in the replicated genome gene in order to insert the genome gene into the shell.

[0063] The MS2 system, which is used for visualization of intracellular RNA, is composed of the above-mentioned MS2 coat protein and loop RNA, and the MS2 coat protein and loop RNA are specifically and strongly linked together within the cell.

[0064] The guide RNA may be a fusion RNA of an MS2 loop RNA and an RNA complementary to at least a portion of a polynucleotide sequence containing guanosine, which is closely related to the pathology of a disease, or a guanosine generated in the polynucleotide by an A-to-G mutation. If the nucleic acid base to be converted is referred to as a "target nucleic acid base," the base sequence of the guide RNA may be composed of a base sequence complementary to at least a portion of the base sequence containing the target nucleic acid base, guanosine, in the polynucleotide. As long as the guide RNA specifically hybridizes to the complementary base sequence, the guide RNA may have a base sequence containing a mismatch with the complementary base sequence, i.e., a base sequence complementary to a base sequence obtained by removing one or several bases from the complementary base sequence. When the guide RNA contains a mismatch, it is preferable that only the nucleic acid base in the guide RNA corresponding to the target nucleic acid base is mismatched. In this case, the nucleic acid base in the guide RNA corresponding to the target nucleic acid base, guanosine, may be a base other than cytosine (C), which forms a Watson-Crick base pair with guanine.

[0065] Fusion RNA of a guide RNA and an MS2 loop RNA (guide RNA-MS2 RNA) can be prepared based on the base sequence of the guide RNA. For example, the fusion RNA may be one in which the 3' end (or 5' end) of the guide RNA is directly linked to one end of the MS2 loop RNA, or one indirectly linked via a linker sequence or the like. The fusion RNA may be one in which the MS2 loop RNA is linked to both ends of the guide RNA.

[0066] The enzyme may be a fusion protein with the MS2 coat protein (MS2 coat protein-enzyme). The fusion protein can bind specifically and strongly to the guide RNA via the bond between the MS2 coat protein and the MS2 loop RNA. For example, the fusion protein may be one in which the carboxyl terminus (or amino terminus) of the MS2 coat protein is directly bound to the amino terminus (or carboxyl terminus) of the enzyme, or one indirectly bound via a linker peptide or the like. The fusion protein can be produced based on the gene sequences of the MS2 coat protein and the enzyme.

[0067] The MS2 loop RNA and the MS2 coat protein have strong binding affinity. Therefore, by mixing the fusion protein and the fusion RNA or by co-expressing them in cells, an enzyme complex composed of "guide RNA-MS2 loop RNA-MS2 protein-enzyme" can be obtained. This enzyme complex complementarily binds to the polynucleotide targeted by the guide RNA in animal cells. For example, the active site of the enzyme can be induced by a guanosine closely related to the pathology of a disease or a guanosine generated in a polynucleotide by an A-to-G mutation, thereby efficiently converting the target nucleic acid base.

[0068] An enzyme complex according to one embodiment of the present invention uses a base sequence recognition module to allow the enzyme (e.g., an enzyme that converts guanosine to xanthosine) to act specifically on a target guanosine. Therefore, by targeting a guanosine closely related to the pathology of a disease or a guanosine resulting from adenosine-to-guanosine mutation, it is possible to convert the guanosine closely related to the pathology and / or repair the genetic code of a mutated polynucleotide. For example, the enzyme complex can convert a guanosine in a polynucleotide resulting from adenosine-to-guanosine mutation into a genetic code similar to adenosine. Furthermore, the enzyme complex can disrupt the guanine-quadruplex structure of TERRA, which has a telomere sequence, by converting the guanosine in the guanine-quadruplex to xanthosine. These conversions can improve the pathology of a disease.

[0069] In another embodiment, a polynucleotide is provided that encodes a complex comprising the fusion protein and the fusion RNA.

[0070] When the base sequence recognition module comprises a nucleic acid that binds to a polynucleotide in a sequence-specific manner, the nucleic acid may be RNA or DNA.In addition, the base sequence recognized by the nucleic acid may be near the target nucleic acid base, and the nucleic acid may or may not contain the target nucleic acid base.In addition, when the base sequence recognition module comprises a protein that binds to a polynucleotide in a sequence-specific manner, the base sequence in the polynucleotide that the protein recognizes may be near the target nucleic acid base, and the base sequence may or may not contain the target nucleic acid base.

[0071] As long as the guide RNA binds to the target polynucleotide in a sequence-specific manner, a mutant of the MS2 coat protein or CRISPR-dCas protein may be used. Furthermore, known polynucleotide-binding proteins and polynucleotides, such as those of the λN system, may be used as the base sequence recognition module. The λN system utilizes the λN22 peptide and the base sequence (Box-B) of the RNA to which the λN22 peptide specifically binds. As long as the mutant exhibits specific binding to the target, a mutant of the λN22 peptide may also be used.

[0072] The polynucleotide-binding protein may also be a derivative of the aforementioned PPR protein or TAL effector, etc. The RNA-binding protein may be, for example, fragile X mental retardation syndrome-related protein 1 (FMRR1), other known sequence-specific polynucleotide-binding proteins, fragments thereof, or derivatives thereof.

[0073] [3. Expression Vector] An expression vector according to one embodiment of the present invention is an expression vector for animals, which comprises a polynucleotide encoding the enzyme complex according to one embodiment of the present invention.

[0074] The expression vector may be one that expresses the enzyme complex in an integrated state within an animal cell, or one that expresses the enzyme and the base sequence recognition module separately within an animal cell and then complexes them together.

[0075] In detail, the expression vector may be one that expresses a fusion protein of the enzyme and the base sequence recognition module in an animal cell, or one that expresses a fusion RNA that is the base sequence recognition module and a fusion protein of the enzyme and the RNA-binding protein in a cell, and then forms a complex by binding the two in the animal cell via specific binding between the fusion RNA and the RNA-binding protein.

[0076] The expression vector may contain a polynucleotide encoding a fusion protein of the enzyme and a protein that is a base sequence recognition module, or may contain, in a single vector, a first polynucleotide encoding an RNA that recognizes a sequence and an RNA that specifically binds to an RNA-binding protein, and a second polynucleotide encoding a fusion protein of the enzyme and the RNA-binding protein, or may contain the first polynucleotide and the second polynucleotide in separate vectors.

[0077] The base sequences of the first polynucleotide and the second polynucleotide can be determined based on the amino acid sequence they encode or the base sequence of the RNA they encode. The first polynucleotide and the second polynucleotide can be prepared based on the base sequences according to known methods. The first polynucleotide and the second polynucleotide can be introduced into various vectors using restriction enzymes, DNA ligase, or a plasmid construction kit based on homologous recombination.

[0078] An intervening linker amino acid sequence may be present between the enzyme and the RNA-binding protein, and an intervening nucleic acid linker sequence may be present between the guide RNA and the RNA that specifically binds to the RNA-binding protein.

[0079] In order to express an enzyme complex in animal cells, an expression vector according to one embodiment of the present invention preferably includes a promoter upstream of a polynucleotide encoding the enzyme complex for controlling the expression of the polynucleotide. Examples of such promoters include CMV, CMV-IE, EF1α, and U6. Among these promoters, EF1α is preferred for polypeptide expression, and U6 is preferred for guide RNA expression, due to the advantages of stable and high expression.

[0080] The form of the expression vector according to one embodiment of the present invention is not limited as long as it can express the enzyme complex according to one embodiment of the present invention. The expression vector according to one embodiment of the present invention may be a DNA type, an RNA type, a circular type, a linear type, or a viral vector. If the expression vector is constructed using DNA, which is more stable than RNA, it can be more stable during storage, etc., than the enzyme complex containing the above-mentioned guide RNA, and it can be handled more easily during use.

[0081] According to an expression vector of one embodiment of the present invention, a complex can be formed in an animal cell by expressing the enzyme and the base sequence recognition module as a fusion protein, or by expressing the enzyme and the base sequence recognition module separately and then self-assembling them via an RNA-binding protein or the like. This allows the enzyme of one embodiment of the present invention to act specifically on its target guanosine in a polynucleotide in the animal cell. Using this expression vector, guanosine, which is closely related to the pathology of a disease, or guanosine generated in a polynucleotide by A to G mutation, can be converted to adenosine. According to an expression vector of one embodiment of the present invention, the enzyme complex can be expressed or formed in an animal cell.

[0082] 4. Genetic Disease Therapeutic Drug A genetic disease therapeutic drug for animals according to one embodiment of the present invention comprises the enzyme complex according to one embodiment of the present invention or the expression vector according to one embodiment of the present invention.

[0083] The genetic disease may be a disease in which guanosine is involved in the cause or aggravation of the pathology, or may be a disease caused by a mutation from adenosine to guanosine.

[0084] The genetic disease therapeutic drug can be produced according to known methods. The genetic disease therapeutic drug may contain other pharmacologically acceptable ingredients in addition to the enzyme complex or expression vector as an active ingredient. The genetic disease therapeutic drug may contain, for example, the enzyme complex or expression vector and a pharmacologically acceptable carrier. The pharmacologically acceptable carrier may be any of various organic or inorganic carrier substances used as formulation materials. Examples of pharmacologically acceptable carriers include (i) excipients, lubricants, binders, and / or disintegrants in solid formulations, and (ii) solvents, solubilizers, suspending agents, isotonicity agents, buffers, and / or soothing agents in liquid formulations. The genetic disease therapeutic drug may also contain additives such as preservatives, antioxidants, colorants, and / or sweeteners, as needed.

[0085] The dosage of the genetic disease therapeutic drug can be determined appropriately depending on the gender, age, weight, and / or symptoms of the subject. In the genetic disease therapeutic drug, the enzyme complex or expression vector is administered to the subject in an effective amount. An effective amount is the amount of the enzyme complex or expression vector required to achieve the desired result, i.e., the amount required to delay, inhibit, prevent, reverse, or cure the condition (symptom) being treated or treated.

[0086] The route of administration of the genetic disease therapeutic agent is not particularly limited, and the genetic disease therapeutic agent is preferably used as an external preparation, an injection, an inhalant, or an oral preparation.

[0087] The above-mentioned genetic disease therapeutic drug can convert guanosine in a polynucleotide that is closely related to the pathology of a disease and / or can convert mutated guanosine in a disease caused by an A to G mutation into a genetic code similar to adenosine by specifically acting an enzyme according to one embodiment of the present invention on a target nucleic acid base in a polynucleotide. Therefore, the above-mentioned genetic disease therapeutic drug is effective in treating diseases caused by gene mutations (e.g., point mutations).

[0088] In another aspect of the present invention, a genetic disease in a subject can be treated, improved, or prevented by administering the enzyme conjugate or the expression vector to the subject. For example, another aspect of the present invention is a "method for treating a genetic disease (or a method for improving or preventing a genetic disease), comprising the step of administering an enzyme conjugate according to one embodiment of the present invention or an expression vector according to one embodiment of the present invention to an animal (e.g., a human or a non-human animal)."

[0089] Another aspect of the present invention is use of the enzyme complex or expression vector described above for producing a drug for treating a genetic disease. For example, another aspect of the present invention is "use of an enzyme complex according to one embodiment of the present invention or an expression vector according to one embodiment of the present invention for producing a drug for treating a genetic disease (or a drug for improving a genetic disease or a drug for preventing a genetic disease)."

[0090] The enzyme complex and / or the expression vector may be used as a reagent for experiments in vitro, in vivo, or ex vivo.

[0091] [5. Others] The invention according to one aspect of the present invention includes the following inventions.

[0092] <1> An enzyme that has the activity of deaminating guanosine in a polynucleotide and converting it to xanthosine in human cells.

[0093] <2> An enzyme that converts the genetic code of guanosine in a polynucleotide into adenosine (the genetic code of adenosine) in human cells.

[0094] <3> The enzyme according to <1> or <2>, comprising: (i) a region having an amino acid sequence shown in SEQ ID NO: 1 or 2; or (ii) a region having an amino acid sequence having 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2.

[0095] <4> An enzyme complex comprising the enzyme according to any one of <1> to <3> and a base sequence recognition module for allowing the enzyme to act specifically on guanosine in a polynucleotide that causes a pathological condition or is involved in the aggravation of the pathological condition.

[0096] <5> An enzyme complex comprising the enzyme according to any one of <1> to <3> and a base sequence recognition module for allowing the enzyme to act specifically on a guanosine generated in a polynucleotide by mutation from A to G.

[0097] <6> An expression vector for expressing and forming the enzyme complex according to <4> or <5> in human cells.

[0098] <7> A therapeutic agent for genetic diseases, comprising the enzyme complex according to <4> or <5>, or the expression vector according to <6>.

[0099] [1] An enzyme having the activity of converting guanosine in a polynucleotide into a genetic code corresponding to adenosine in an animal cell.

[0100] [2] The enzyme according to [1], comprising any one of the following polypeptides (1) to (6): (1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2; (2) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2 in which one or more amino acids have been substituted, deleted, inserted, and / or added; (3) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2; (4) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 4; (5) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 4; (6) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 3 or 4.

[0101] [3] The enzyme according to [1] or [2], further comprising an E2 domain which is any one of the polypeptides (7) to (12): (7) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5; (8) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 5; (9) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 5; (10) a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6; (11) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide having a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6; (12) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 6.

[0102] [4] An enzyme complex comprising the enzyme according to any one of [1] to [3] and a base sequence recognition module that binds to a specific sequence in a polynucleotide, thereby allowing the enzyme to act on a specific guanosine in the polynucleotide sequence.

[0103] [5] The enzyme complex according to [4], wherein the base sequence recognition module (a) binds to a specific sequence in a polynucleotide involved in the cause of a pathology or the aggravation of the pathology, and / or (b) binds to a specific sequence in a polynucleotide having a mutation in which adenosine is substituted with guanosine.

[0104] [6] An expression vector for animals, comprising a polynucleotide encoding the enzyme complex according to [4] or [5].

[0105] [7] A therapeutic drug for genetic diseases for animals, comprising the enzyme complex according to [4] or [5], or the expression vector according to [6].

[0106] [8] A method for treating a genetic disease, comprising the step of administering the genetic disease therapeutic drug for animals according to [7] to a subject (e.g., a human or a non-human animal).

[0107] [9] Use of the enzyme complex according to [4] or [5] or the expression vector according to [6] for the production of a genetic disease therapeutic drug for animals.

[0108] The above describes one embodiment of the present invention. One embodiment of the present invention may also contribute to the achievement of Goal 3 of the Sustainable Development Goals (SDGs) advocated by the United Nations, which is to "ensure good health and well-being for all."

[0109] The following examples will explain one embodiment of the present invention in more detail, but the present invention is not limited to these examples.

[0110] <1. Detection of polynucleotide editing activity in animal cells> <1-1. Test method> A plasmid was prepared for expressing the "PPR56-E2-DRH" fusion protein in animal cells. The specific structure of the "PPR56-E2-DRH" fusion protein was such that the E2 domain was directly linked to the carboxyl terminus of the PPR domain of the PPR56 protein of Physcomitrium patens (NCBI identification number: LOC112295756), and the DRH domain was further linked to the carboxyl terminus of the E2 domain.

[0111] Specifically, the amino acid sequence of the "PPR56-E2-DRH" fusion protein was such that, from the amino terminus, the amino acid sequence of SEQ ID NO: 7, the amino acid sequence of SEQ ID NO: 5, and the amino acid sequence of SEQ ID NO: 1 were linked together without a linker. On the other hand, the nucleotide sequence of the "PPR56-E2-DRH" fusion protein was such that, from the 5' terminus, the nucleotide sequence of SEQ ID NO: 8, the nucleotide sequence of SEQ ID NO: 6, and the nucleotide sequence of SEQ ID NO: 3 were linked together without a linker. In this plasmid, PPR56 is the sequence recognition module, and E2-DRH exhibits enzymatic activity.

[0112] DNA encoding the "PPR56-E2-DRH" fusion protein was inserted into a plasmid (pSC101), and a CMV promoter for expressing the "PPR56-E2-DRH" fusion protein in animal cells was inserted upstream of the DNA. The plasmid thus constructed was designated pC101-PPR56-E2-DRH (see Figure 1).

[0113] As shown in Figure 2, the PPR domain of the PPR56 protein recognizes a polynucleotide having the base sequence "UAUAGACGGUAUCUCUCU" (SEQ ID NO: 9) in mRNA transcribed from the Nad4 gene and specifically binds to the polynucleotide. Therefore, the "E2-DRH" domain in the "PPR56-E2-DRH" fusion protein can access the "G" present at the 3' end of "UAUAGACGGUAUCUCUCU".

[0114] The target gene used in this test was the EGFP gene in which the initiation codon "ATG" had been converted to "GTG," and DNA corresponding to the PPR56 recognition base sequence at the 5' end of the EGFP gene had been inserted.

[0115] In the target gene, the initiation codon has been lost due to mutation, so that the EGFP protein cannot be biosynthesized normally and the fluorescence derived from the EGFP cannot be emitted.

[0116] On the other hand, if the first "G" of "GTG" resulting from the mutation is converted to xanthosine by deamination, the xanthosine will be recognized as "A." In other words, if the first "G" of "GTG" resulting from the mutation is converted to xanthosine by deamination, the "GTG" will be synonymous with the start codon "ATG" in the genetic code. As a result, the EGFP protein is biosynthesized, and fluorescence derived from the EGFP will be emitted.

[0117] HEK293 cells, a human cell line, were plated at 6.4 × 10 ５Before transfection, the culture medium in the glass-bottom dish was removed, and 2 mL of fresh FBS-containing DMEM was added to the glass-bottom dish.

[0118] Next, (i) a tube containing 100 μL of Opti-MEM and 4 μL of Lipofectamine 3000 reagent, and (ii) a tube containing 100 μL of Opti-MEM and 4 μL of a plasmid solution ((a) a mixture of 1600 ng of pC101-PPR56-E2-DRH and 400 ng of mutated EGFP plasmid, or (b) 400 ng of mutated EGFP plasmid alone) were prepared. The solutions in the two tubes were mixed and incubated at room temperature for 10 minutes, after which the mixed solution was added to a glass-bottom dish. 24 hours after transfection, green fluorescence was observed using a confocal laser microscope to confirm whether or not it had returned to normal.

[0119] <2-2. Test Results> The test results are shown in Figure 3. Images 301 and 302 in Figure 3 are images of human cells into which only the mutated EGFP plasmid was introduced, and images 303 and 304 in Figure 3 are images of human cells into which the mutated EGFP plasmid and pC101-PPR56-E2-DRH were introduced, respectively.

[0120] As is clear from 302 in Figure 3, fluorescence derived from EGFP was not observed in human cells into which only the mutated EGFP plasmid was introduced. On the other hand, as is clear from 304 in Figure 3, fluorescence derived from EGFP was observed in human cells into which the mutated EGFP plasmid and pC101-PPR56-E2-DRH were introduced. This indicates that the first "G" of "GTG" generated by the mutation was deaminated and converted to xanthosine by the "DRH domain," and that the xanthosine was recognized as "A," in other words, that "GTG" was converted and repaired to the start codon "ATG."

[0121] The present invention can be used in medicines or research reagents.

Claims

1. An enzyme that converts guanosine in a polynucleotide into the genetic code equivalent to adenosine in animal cells.

2. The enzyme according to claim 1, comprising any one of the following polypeptides (1) to (6): (1) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2; (2) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2 in which one or more amino acids have been substituted, deleted, inserted, and / or added; (3) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2; (4) a polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 3 or 4; (5) a polypeptide encoded by a polynucleotide that hybridizes under stringent conditions to a polynucleotide consisting of a base sequence complementary to the polynucleotide consisting of the base sequence of SEQ ID NO: 3 or 4; (6) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO: 3 or 4.

3. The enzyme described in claim 2, further comprising an E2 domain which is any one of the polypeptides (7) to (12): (7) a polypeptide consisting of the amino acid sequence of SEQ ID NO:5; (8) a polypeptide consisting of an amino acid sequence in which one or more amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO:5; (9) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO:5; (10) a polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO:6; (11) a polypeptide encoded by a polynucleotide which hybridizes under stringent conditions to a polynucleotide consisting of a base sequence complementary to the polynucleotide consisting of the base sequence of SEQ ID NO:6; (12) a polypeptide consisting of an amino acid sequence having 90% or more sequence identity with the amino acid sequence of a polypeptide encoded by a polynucleotide consisting of the base sequence of SEQ ID NO:

6.

4. An enzyme complex comprising the enzyme according to claim 1 and a base sequence recognition module that binds to a specific sequence within a polynucleotide, thereby allowing the enzyme to act on a specific guanosine within the polynucleotide sequence.

5. The enzyme complex described in claim 4, wherein the base sequence recognition module (a) binds to a specific sequence in a polynucleotide involved in the cause or aggravation of a pathology, and / or (b) binds to a specific sequence in a polynucleotide having a mutation in which adenosine is replaced with guanosine.

6. An expression vector for animals, comprising a polynucleotide encoding the enzyme complex according to claim 4.

7. A genetic disease therapeutic agent for animals, comprising the enzyme complex according to claim 4 or the expression vector according to claim 6.

Images