Method for controlling translation amount of target protein

Abstract

Provided is a method for controlling the translation amount of a target protein based on target RNA that encodes the target protein. The method for controlling the translation amount of a target protein includes editing a base sequence represented by AUA or AUG included in a 5' untranslated region of mRNA including a coding region of a target protein into a base sequence represented by AUI or IUG in the presence of adenosine deaminase. The method may include: editing at least one of the base sequences represented by AUG included in the 5' untranslated region of the mRNA serving as a target into a base sequence represented by IUG; and setting the number of base sequences represented by AUG included in the 5' untranslated region after editing to 1 or less. The method may also include editing at least one of the base sequences represented by AUA included in the 5' untranslated region of the mRNA serving as a target into a base sequence represented by AUI, and the number of base sequences represented by AUG included in the 5' untranslated region may be 0.

Description

Method for controlling the translation amount of a target protein

[0001] The present invention relates to a method for controlling the translation amount of a target protein.

[0002] With the development of genome editing technology, methods for controlling life phenomena by modifying genetic information, the blueprint of living organisms, i.e., DNA information within cells, are beginning to be used in medicine and drug discovery as a disease treatment approach. Because DNA is a constant and unchanging molecule within cells, the effects of DNA modification remain permanently in the target cell or organism. On the other hand, RNA is a nucleic acid molecule that contains copied DNA information, and unlike DNA, it is a transient genetic information molecule that undergoes repeated synthesis and decomposition. Therefore, modifying RNA information can impart a temporary, non-permanent genetic information modification effect to the target organism. In other words, although RNA modification technology is a genetic modification technology like DNA modification, its properties are significantly different.

[0003] As an RNA modification technique, for example, WO 2017 / 010556 describes a site-specific RNA mutagenesis method in which double-strand-specific adenosine deaminase (ADAR) acts on a complex between a target RNA and a target-editing guide RNA. Also, WO 2022 / 124345 describes a pharmaceutical composition for treating a disease by converting adenosine residues in a target RNA to inosine residues.

[0004] An object of one aspect of the present invention is to provide a method for controlling the translation amount of a target protein based on a target RNA that encodes the target protein.

[0005] The first aspect is a method for controlling the translation amount of a target protein, which comprises editing a base sequence represented by AUA or AUG contained in the 5' untranslated region (hereinafter sometimes abbreviated as 5'-UTR) of mRNA containing the coding region of the target protein into a base sequence represented by AUI or IUG in the presence of adenosine deaminase (hereinafter sometimes abbreviated as ADAR).

[0006] The second aspect is a method for controlling the translation amount of a target protein, comprising contacting, in the presence of adenosine deaminase, mRNA comprising a 5' untranslated region containing a base sequence represented by AUA or AUG and a coding region for a target protein with a target editing guide oligonucleotide capable of editing the base sequence represented by AUA or AUG contained in the 5' untranslated region into a base sequence represented by AUI or IUG in the presence of adenosine deaminase.

[0007] The method for controlling the translation amount of a target protein may be a method for enhancing the translation amount of a target protein. The method for enhancing the translation amount of a target protein may include editing at least one base sequence represented by AUG contained in the 5' untranslated region of a target mRNA to a base sequence represented by IUG, and may include reducing the number of base sequences represented by AUG contained in the edited 5' untranslated region to 1 or less, or to 0.

[0008] The method for controlling the translation amount of a target protein may be a method for suppressing the translation amount of a target protein. The method for suppressing the translation amount of a target protein may include editing at least one nucleotide sequence represented by AUA contained in the 5' untranslated region of a target mRNA to a nucleotide sequence represented by AUI, and the number of nucleotide sequences represented by AUG contained in the 5' untranslated region may be zero.

[0009] In the method for controlling the translation amount of a target protein, the 5' untranslated region of the target mRNA may contain 6 to 1200 ribonucleotide residues, and the base sequence represented by AUA or AUG may be located 4 to 1200 residues from the 5' capping of the mRNA.

[0010] A third aspect is a method for controlling the translation amount of a target protein, comprising editing a base sequence represented by AUG, which is the start codon of mRNA containing the coding region of the target protein, to a base sequence represented by IUG in the presence of adenosine deaminase. The method for controlling the translation amount of a target protein may be a method for suppressing the translation amount of the target protein, and may comprise contacting, in the presence of adenosine deaminase, mRNA containing the coding region of the target protein with a target editing guide oligonucleotide that can edit the base sequence represented by AUG, which is the start codon of the target protein, to a base sequence represented by IUG.

[0011] According to one aspect of the present invention, a method for controlling the translation amount of a target protein based on a target RNA encoding the target protein can be provided.

[0012] FIG. 1 is a schematic diagram showing the structure of the reporter RNA used in Test Example 1. FIG. 2 is a diagram showing the results of editing analysis in Test Example 1. FIG. 3 is a diagram showing the results of luciferase reporter assay in Test Example 1. FIG. 4 is a diagram showing the results of luciferase reporter assay in Test Example 1. FIG. 5 is a schematic diagram showing the structure of the reporter RNA used in Test Example 2. FIG. 6 is a diagram showing the results of editing analysis in Test Example 2. FIG. 7 is a diagram showing the results of luciferase reporter assay in Test Example 2. FIG. 8 is a schematic diagram showing the structure of the reporter RNA used in Test Example 3. FIG. 9 is a diagram showing the results of luciferase reporter assay in Test Example 3. FIG. 10 is a diagram showing the results of editing analysis in Test Example 4. FIG. 11 is a diagram showing the results of luciferase reporter assay in Test Example 4. FIG. 12 is a diagram showing the results of luciferase reporter assay in Test Example 4. FIG. 13 is a diagram showing the results of editing analysis in Test Example 5. FIG. 14 is a diagram showing the results of luciferase reporter assay in Test Example 5. FIG. 15 is a diagram showing the results of luciferase reporter assay in Test Example 5. FIG. 16 is a schematic diagram showing an example of the structure of the reporter RNA used in Test Example 7. FIG. 17 is a schematic diagram showing another example of the structure of the reporter RNA used in Test Example 7. 1 shows the results of editing analysis in Test Example 7. FIG. 2 shows the results of luciferase reporter assay in Test Example 7. FIG. 3 shows the results of luciferase reporter assay in Test Example 7.

[0013] As used herein, the term "step" refers not only to an independent step, but also to a step that cannot be clearly distinguished from other steps, as long as the intended purpose of the step is achieved. Furthermore, the upper and lower limits of the numerical ranges described herein can be arbitrarily selected and combined from the numerical values ​​exemplified as numerical ranges. Hereinafter, embodiments of the present invention will be described in detail. However, the embodiments described below exemplify methods for controlling the translation amount of a target protein in order to embody the technical concept of the present invention, and the present invention is not limited to the methods for controlling the translation amount of a target protein described below.

[0014] Method for controlling the translation amount of a target protein A method for controlling the translation amount of a target protein includes editing a base sequence represented by AUA or AUG contained in the 5' untranslated region of an mRNA containing a coding region for the target protein to a base sequence represented by AUI or IUG in the presence of adenosine deaminase (ADAR). One aspect of the method for controlling the translation amount of a target protein may include contacting, in the presence of ADAR, an mRNA containing a 5' untranslated region containing a base sequence represented by AUA or AUG and a coding region for the target protein with a target editing guide oligonucleotide that can edit the base sequence represented by AUA or AUG contained in the 5' untranslated region to a base sequence represented by AUI or IUG in the presence of ADAR. Note that in the method for controlling the translation amount of a target protein, in the presence of ADAR, the base sequence represented by AUA is edited to a base sequence represented by AUI, and the base sequence represented by AUG is edited to a base sequence represented by IUG.

[0015] ADAR is an enzyme that converts adenosine residues targeted in double-stranded RNA into inosine residues through hydrolytic deamination, and is widely present in mammalian cells. Inosine residues are similar in structure to guanosine residues, and are therefore translated as guanosine residues during translation of RNA information. For example, when the third adenosine residue from the 5' end of a base sequence represented by AUA in mRNA (hereinafter also referred to as the AUA sequence) is edited by ADAR to become an inosine residue, the resulting base sequence is represented by AUI, which can be recognized as the start codon AUG by the ribosome, thereby initiating protein translation starting from that start codon. Furthermore, when an adenosine residue in a base sequence represented by AUG in mRNA (hereinafter also referred to as the AUG sequence) is edited by ADAR to become an inosine residue, the resulting base sequence is represented by IUG, which is no longer recognized as the start codon AUG, and protein translation starting from that start codon is not initiated.

[0016] In one aspect of the present invention, the translational amount of the target protein can be suppressed by editing a base sequence represented by AUA contained in the 5' untranslated region of mRNA containing the coding region of the target protein into a base sequence represented by AUI in the presence of adenosine deaminase. The base sequence represented by AUI edited by the action of adenosine deaminase is recognized by the ribosome as an initiation codon represented by AUG, and translation begins from the 5' untranslated region that is not normally translated. This reduces the translation efficiency of the target protein, resulting in suppression of the translational amount of the target protein.

[0017] In one aspect of the present invention, the translation amount of the target protein can be increased by editing a base sequence represented by AUG contained in the 5' untranslated region of mRNA containing the coding region of the target protein to a base sequence represented by IUG in the presence of adenosine deaminase. If a base sequence represented by AUG is present in the 5' untranslated region of mRNA, it can be recognized as a potential start codon, and the translation amount of the target protein from the start codon is controlled to a reduced state. When the base sequence AUG that can be recognized as a start codon in the 5' untranslated region is edited by ADAR to a base sequence represented by IUG and is no longer recognized as a start codon, the translation efficiency from the start codon of the target protein increases, resulting in an increase in the translation amount of the target protein.

[0018] In another aspect, the present invention may include a method for controlling the translation amount of a target protein, comprising editing a base sequence represented by AUG, which is the start codon of the target protein in mRNA encoding the target protein, to a base sequence represented by IUG in the presence of ADAR. The start codon of the target protein is edited by ADAR to a base sequence represented by IUG, so that it is no longer recognized as an initiation codon, thereby suppressing the translation amount of the target protein.

[0019] In one aspect of the present invention, the double-stranded RNA recognized by ADAR is formed, for example, from an mRNA to be edited and a target editing guide oligonucleotide that specifies the editing position on the mRNA. The mRNA to be edited by ADAR may be a mature mRNA formed through a series of processing steps, in addition to a pre-mRNA immediately after transcription. The mature mRNA may include a 5' cap added to the 5' end, a 5' untranslated region located before the start codon of the target protein and containing a base sequence represented by AUA or AUG, a coding region for the target protein from the start codon to the stop codon, a 3' untranslated region located after the stop codon, and a 3' poly(A) tail. The mRNA to be edited may be a monocistronic type that translates a single protein, or a polycistronic type that can translate multiple proteins.

[0020] The mRNA to be edited may contain a base sequence represented by AUG or AUA in its 5' untranslated region, and the target protein translated from the coding region is not particularly limited. mRNAs that contain a base sequence represented by AUG or AUA in their 5' untranslated region and can be edited can be selected by searching a genome information database. For example, a search of the genome database Ensembl reveals that there are more than 50,000 transcripts that code for proteins and contain an AUG sequence in their 5' untranslated region, and even if we limit it to transcripts with only one AUG sequence in their 5' untranslated region, there are more than 17,000. In other words, the method for controlling the translation rate of a target protein according to this embodiment has an extremely wide range of application. For example, since the mRNA of GATA4, a zinc finger transcription factor, contains a single AUG sequence in its 5' untranslated region, it is believed that the translation rate can be increased by RNA editing. On the other hand, since the mRNA of UDP-glucose pyrophosphorylase (UGP2) contains an AUA sequence in its 5' untranslated region, it is believed that the translation rate can be suppressed by RNA editing.

[0021] The 5' untranslated region of the mRNA to be edited need only contain a base sequence represented by AUG or AUA, and the number of residues is not particularly limited. The number of residues in the 5' untranslated region may be, for example, 6 to 1200 or 6 to 1194, preferably 6 to 400. The position of the base sequence represented by AUG or AUA in the 5' untranslated region is not particularly limited. The base sequence represented by AUG or AUA in the 5' untranslated region may be located, for example, from 4 to 1200 residues or 4 to 1194 residues from the 5' capping of the mRNA, preferably from 6 to 200 residues. Note that the position of 4 residues from the 5' capping is specified by considering the nucleotide residue to which the 5' capping is attached as the first residue.

[0022] When the mRNA to be edited contains a base sequence represented by AUG in its 5' untranslated region, the number of base sequences represented by AUG contained in the 5' untranslated region may be, for example, 1 to 3, and preferably 1 to 2. Furthermore, when the mRNA to be edited contains a base sequence represented by AUA in its 5' untranslated region, the number of base sequences represented by AUA contained in the 5' untranslated region may be, for example, 1 or more, and preferably 10 or less, or 8 or less.

[0023] The mRNA to be edited may not contain a base sequence represented by AUG or AUA in the 5' untranslated region. In this case, the AUG sequence, which is the initiation codon of the target protein, becomes the editing target.

[0024] The target editing guide oligonucleotide (hereinafter sometimes abbreviated as gRNA) can form a double-stranded RNA recognizable by ADAR together with the mRNA to be edited, and can make the adenosine residue that is the editing target on the mRNA editable by ADAR. The base sequence of the target editing guide oligonucleotide can be designed according to the base sequence of the 5' untranslated region of the mRNA to be edited, for example, with reference to the descriptions in International Publication No. 2017 / 010556, International Publication No. 2019 / 111957, International Publication No. 2021 / 060527, Nat. Biotechnol. 40, 1093. (2022). The target editing guide oligonucleotide may have a base sequence that can edit a base sequence represented by AUA contained in the 5' untranslated region of the mRNA to be edited into a base sequence represented by AUI in the presence of ADAR, or a base sequence that can edit a base sequence represented by AUG contained in the 5' untranslated region of the mRNA to be edited into a base sequence represented by IUG in the presence of ADAR. The target editing guide oligonucleotide may preferably have the configuration of any of the first to third target editing guide oligonucleotides exemplified below.

[0025] The first target editing guide oligonucleotide may include a first oligonucleotide that forms a complementary strand with the mRNA to be edited and a second oligonucleotide linked to the 5' end of the first oligonucleotide. The first oligonucleotide may have a base sequence consisting of a target-corresponding nucleotide residue corresponding to the third adenosine residue from the 5' end of the base sequence represented by AUA in the 5' untranslated region of the mRNA, or an adenosine residue in the base sequence represented by AUG, a 10- to 24-residue oligonucleotide linked to the 3' end of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, and a 3- to 6-residue oligonucleotide linked to the 5' end of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA. The second oligonucleotide may be an 8- to 50-residue oligonucleotide and may have a base sequence capable of forming a stem-loop structure within the molecule.

[0026] The second target editing guide oligonucleotide may include a third oligonucleotide that forms a complementary strand with the mRNA to be edited, and a fourth oligonucleotide linked to the 3' end of the third oligonucleotide. The third oligonucleotide may have a base sequence consisting of a target-corresponding nucleotide residue corresponding to the third adenosine residue from the 3' end of the base sequence represented by AUA in the 5' untranslated region of the mRNA, or an adenosine residue in the base sequence represented by AUG, a 10- to 24-residue oligonucleotide linked to the 5' end of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, and a 3- to 6-residue oligonucleotide linked to the 3' end of the target-corresponding nucleotide residue. The fourth oligonucleotide may be an oligonucleotide of 8 to 50 residues and have a base sequence capable of forming a stem-loop structure within the molecule.

[0027] The third target editing guide oligonucleotide may include a fifth oligonucleotide that forms a complementary strand with the mRNA to be edited and a sixth oligonucleotide linked to the 5' side of the fifth oligonucleotide. The fifth oligonucleotide may have a base sequence consisting of a target-corresponding nucleotide residue corresponding to the third adenosine residue from the 5' side of the base sequence represented by AUA in the 5' untranslated region of the mRNA or an adenosine residue in the base sequence represented by AUG, a 10- to 24-residue oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, and a 3- to 6-residue oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue. The sixth oligonucleotide may lack a nucleotide residue corresponding to the nucleotide residue in the mRNA at its 3' end or may have a nucleotide residue that does not form a complementary pair with the nucleotide residue in the mRNA, and may have 2 to 10 residues, and at least nucleotide residues other than those at the 3' end may form a double-stranded structure complementary to the mRNA.

[0028] For details of the first target editing guide oligonucleotide and the second target editing guide oligonucleotide, reference can be made to the descriptions in, for example, International Publication No. 2017 / 010556 and International Publication No. 2019 / 111957. Specifically, the first target editing guide oligonucleotide may be in the following form. In the first oligonucleotide, the target-corresponding nucleotide residue is a base that does not form a base pair with an adenosine residue and may be a cytidine residue or a derivative thereof. The number of residues of the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue may preferably be 15 to 24, and the number of residues of the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue may preferably be 3. In one embodiment, the number of residues of the second oligonucleotide may be 8 to 50, and may be preferably represented by SEQ ID NO: 1 or 2, and may include a base sequence that can form a stem-loop structure, for example, the base sequence represented by SEQ ID NO: 1.

[0029]

[0030] In another aspect, the number of residues in the second oligonucleotide may be 2 to 24, preferably 20 or less, 16 or less, 15 or less, or 14 or less, and may be 3 or more, 4 or more, 8 or more, 12 or more, or 13 or more. The number of nucleotide residue pairs constituting the stem portion of the second oligonucleotide may be, for example, 2 or more, preferably 3 or more, 4 or more, 5 or more, or 6 or more, and may be 10 or less, 9 or less, 8 or less, or 7 or less. The number of nucleotide residues constituting the loop portion may be, for example, 4 or 5. Specifically, as the base sequence of the second oligonucleotide forming a stem-loop structure, the base sequence of the stem portion preferably contains guanine (G) and cytosine (C) from the viewpoint of the stability of the double-stranded structure. The proportion of G-C pairs among the base pairs in the stem portion may be, for example, 60% or more, or 70% or more. It is also possible to include uracil (U) instead of cytosine (C), which can form a base pair with guanine (G) by tautomerization. The base sequence of the second oligonucleotide preferably includes, in the 5'-side stem portion, at least one selected from the group consisting of a sequence consisting of two or three consecutive guanines (GG or GGG), a sequence consisting of consecutive uracil and guanine (UG), and a sequence consisting of consecutive guanine, uracil, and guanine (GUG), and includes, in the 3'-side stem portion, a sequence capable of forming a complementary pair with the above sequence (e.g., CC, CCC, CCU, CA, CAC). It is also preferable that the 5'-side stem portion includes a sequence consisting of consecutive guanine, uracil, and guanine (GUG) and the 3'-side stem portion includes a sequence capable of forming a complementary pair with the above sequence (e.g., CAC). It is also preferable that the 5'-side stem portion includes a loop portion and a sequence consisting of consecutive guanine, uracil, and guanine (GUG), and includes, in the 3'-side stem portion, a sequence capable of forming a complementary pair with the above sequence (e.g., CAC).

[0031] The second target editing guide oligonucleotide may be in the following form. In the third oligonucleotide, the target-corresponding nucleotide residue is a base that does not form a base pair with an adenosine residue, and may be a cytidine residue or a derivative thereof. The number of residues of the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue may preferably be 15 to 24 residues, and the number of residues of the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue may preferably be 3. In one embodiment, the number of residues of the fourth oligonucleotide may be 8 to 50 residues, and may preferably include the base sequence represented by SEQ ID NO: 1 or 2, for example, the base sequence represented by SEQ ID NO: 2.

[0032] In another embodiment, the number of residues in the fourth oligonucleotide may be 2 to 24, preferably 20 or less, 16 or less, 15 or less, or 14 or less, and may be 3 or more, 4 or more, 8 or more, 12 or more, or 13 or more. The number of nucleotide residue pairs constituting the stem portion of the fourth oligonucleotide may be, for example, 2 or more, preferably 3 or more, 4 or more, 5 or more, or 6 or more, and may be 10 or less, 9 or less, 8 or less, or 7 or less. The number of nucleotide residues constituting the loop portion may be, for example, 4 or 5. Specifically, as the base sequence of the fourth oligonucleotide forming a stem-loop structure, from the viewpoint of the stability of the double-stranded structure, it is preferable that the base sequence of the stem portion contains guanine (G) and cytosine (C). The proportion of G-C pairs among the base pairs in the stem portion may be, for example, 60% or more, or 70% or more. It is to be noted that the second oligonucleotide may contain, instead of cytosine (C), uracil (U), which can form a base pair with guanine (G) by tautomerization. Preferably, the base sequence of the second oligonucleotide contains, in the stem portion on the 3' side, at least one selected from the group consisting of a sequence consisting of two or three consecutive guanines (GG or GGG), a sequence consisting of consecutive uracil and guanine (UG), a sequence consisting of consecutive guanine, uracil and guanine (GUG), and a sequence consisting of consecutive uracil, guanine and guanine (UGG), and contains, in the stem portion on the 5' side, a sequence capable of forming a complementary pair with these (e.g., CC, CCC, CCU, CA, CAC, CCA). It is also preferred that the 3' stem portion contains a sequence consisting of consecutive guanine, uracil and guanine (GUG) or consecutive uracil, guanine and guanine (UGG) with respect to the loop portion, and that the 5' stem portion contains a sequence capable of forming a complementary pair with the sequence (e.g., CAC or CCA).

[0033] For details of the third target editing guide oligonucleotide, reference can be made to the descriptions in, for example, WO 2021 / 060527 and WO 2021 / 124345. Specifically, the third target editing guide oligonucleotide may be as follows. In the fifth oligonucleotide, the target-corresponding nucleotide residue is a base that does not form a base pair with an adenosine residue and may be a cytidine residue or a derivative thereof. The number of residues of the oligonucleotide linked to the 3' side of the target-corresponding nucleotide residue and having a base sequence complementary to the target RNA may preferably be 11 or more, 12 or more, or 13 or more, and may be 22 or less, 20 or less, 18 or less, 16 or less, or 15 or less. The number of residues of the oligonucleotide linked to the 5' side of the target-corresponding nucleotide residue and having a base sequence complementary to the target RNA may preferably be 3 or more and 5 or less, 3 or more and 4 or less, or 3. The number of residues of the sixth oligonucleotide may preferably be 3 or more, 4 or more, or 5 or more, and may be 9 or less, 8 or less, or 7 or less.

[0034] The nucleotide residues constituting the target editing guide oligonucleotide may be natural ribonucleotide residues or non-natural modified nucleotide residues. Modified nucleotide residues include those in which the internucleoside phosphodiester bond has been modified, those in which the 2' hydroxyl group of ribose has been modified, those containing intramolecularly bridged ribose, and those in which at least one of the purine base and pyrimidine base has been modified. Examples of modifications of the phosphodiester bond moiety include phosphorothioate, methylphosphonate, methylthiophosphonate, phosphorodithioate, phosphoramidate, peptide bond substitution, and the like. Examples of modifications of the 2' hydroxyl group of ribose include 2'-O-methylation, 2'-O-methoxyethylation, 2'-O-aminopropyl (AP) modification, 2'-fluorolation, 2'-O-methylcarbamoylethylation, 3,3-dimethylallylation, deoxyribonucleotide modification, and the like. An example of an intramolecular bridge of ribose is a nucleotide bridged at the 2'- and 4'-positions (2',4'-BNA). 2',4'-BNA includes, for example, locked nucleic acids (α-L-methyleneoxy (4'-CH２ -O-2')BNA or β-D-methyleneoxy(4'-CH ２ -O-2')BNA, also known as ENA, ２ ) ２ -O-2')BNA), β-D-thio(4'-CH ２ -S-2')BNA, aminooxy (4'-CH ２ -O-N(R)-2')BNA (R is H or CH ３ ), oxyamino (4'-CH ２ —N(R)—O-2′)BNA (R is H or CH ３ ), 2',4'-BNACOC, 3'-amino-2',4'-BNA, 5'-methylBNA, cEt-BNA (4'-CH(CH ３ )-O-2')BNA, also called cMOE-BNA (4'-CH(CH ２ OCH ３ )-O-2') BNA, amide type BNA also called AmNA (4'-C(O)-N(R)-2') BNA (R is H or CH ３ Examples of modifications of the base moiety include halogenation; alkylation such as methylation, ethylation, n-propylation, isopropylation, cyclopropylation, n-butylation, isobutylation, s-butylation, t-butylation, and cyclobutylation; hydroxylation; amination; deamination; demethylation; and the like.

[0035] As adenosine deaminase (ADAR) having RNA editing activity, ADAR1 and ADAR2 isoforms are known. These are thought to be expressed in a cell type-specific manner in vivo. For example, ADAR1 is thought to be hardly expressed in human skeletal muscle cells. On the other hand, ADAR2 is thought to be hardly expressed in human bone marrow cells. In addition, ADAR1 is generally thought to be more highly expressed in human cells. The ADAR used in the method for controlling the translation amount of a target protein may be ADAR1 or ADAR2. Preferably, it may be ADAR1.

[0036] The ADAR used in the method for controlling the translation amount of a target protein may be an ADAR present in a cell, or an ADAR introduced from outside the cell. The introduction of the ADAR from outside the cell can be carried out by a conventional method. For example, the desired ADAR can be introduced into a cell by constructing a vector that expresses the desired ADAR by a conventional method and introducing it into the cell by an appropriate method.

[0037] A method for controlling the translational amount of a target protein includes contacting an mRNA to be edited with a target editing guide oligonucleotide in the presence of ADAR. ADAR recognizes the double-stranded RNA formed by the mRNA and the target editing guide oligonucleotide, and edits the base sequence represented by AUA in the 5' untranslated region of the mRNA to a base sequence represented by AUI, thereby suppressing the translational amount of the target protein. Furthermore, ADAR edits the base sequence represented by AUG in the 5' untranslated region of the mRNA to a base sequence represented by IUG, thereby enhancing the translational amount of the target protein. The method for controlling the translational amount of a target protein may be performed in vitro or in vivo.

[0038] The method for controlling the translation amount of a target protein can be carried out, for example, by introducing or expressing the above-mentioned target editing guide oligonucleotide into a eukaryotic cell having the mRNA to be edited. The method for introducing the target editing guide oligonucleotide into a eukaryotic cell can be appropriately selected from various techniques used in nucleic acid medicines. In addition, the target editing guide oligonucleotide can be expressed in the eukaryotic cell by introducing a plasmid or the like capable of expressing the target editing guide oligonucleotide into the eukaryotic cell.

[0039] The method for controlling the translation amount of a target protein may include editing each of a plurality of adenosine residues contained in the 5' untranslated region to inosine residues. Editing of the plurality of adenosine residues may be performed using target editing guide oligonucleotides corresponding to each adenosine residue, or may be performed using target editing guide oligonucleotides capable of simultaneously recognizing multiple adenosine residues that are editing targets.

[0040] One embodiment of the method for controlling the translation amount of a target protein may include editing at least one of the base sequences represented by AUG contained in the 5' untranslated region to a base sequence represented by IUG. When the 5' untranslated region has multiple base sequences represented by AUG, the number of base sequences represented by AUG contained in the edited 5' untranslated region may be, for example, 2 or less, preferably 1 or less, or may be 0.

[0041] One embodiment of the method for controlling the translation level of a target protein may include editing at least one nucleotide sequence represented by AUA contained in the 5' untranslated region to a nucleotide sequence represented by AUI. The 5' untranslated region before editing may further contain a nucleotide sequence represented by AUG in addition to the nucleotide sequence represented by AUA. When the 5' untranslated region before editing contains a nucleotide sequence represented by AUG, the number of nucleotide sequences represented by AUG may be, for example, 1 or less, and preferably 0.

[0042] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0043] Test Example 1: Generation of a start codon and deletion of the start codon by A-to-I RNA editing in a cell-free environment (A) Synthesis of reporter RNA (Nluc rep. AUA, Nluc rep. AUG) Using pcDNA3.1 Hygro(-)_NLuc rep. AUA (or AUG) (SEQ ID NO: 3; XXX is ATA or ATG) as a template, template DNA was prepared by PCR (denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 20 seconds) using T7proGG (SEQ ID NO: 4) and Fluc_Sac_R01 primer (SEQ ID NO: 5) and PrimeStar GXL DNA Polymerase (TaKaRa), and then purified by phenol / chloroform extraction and ethanol precipitation. Reporter RNA was synthesized by in vitro transcription (37°C, 3 hours) using the T7-Scribe Standard RNA IVT KIT (CELLSCRIPT). DNase (final concentration: 2 U) was then added, and the mixture was treated at 37°C for 30 minutes. The RNA was then purified by phenol / chloroform extraction and ethanol precipitation. The resulting reporter RNA was purified by gel filtration (BIO RAD). The structures of the reporter RNAs (Nluc rep. AUA and Nluc rep. AUG) are shown in Figure 1.

[0044]

[0045]

[0046] (B) Target Editing Guide Oligonucleotide (gRNA) Synthesis As transcription template DNA, 2 μM 3ASg_GluNluc_AUI_F01 (SEQ ID NO: 10; FW oligo) and 2 μM 3ASg_GluNluc_AUI_R01 (SEQ ID NO: 11; RV oligo) were added, heated at 95 ° C for 3 minutes, denatured, and then cooled to 25 ° C over 15 minutes for annealing. Then, 0.2 mM dNTP and 2.5 U Klenow fragment (New England Biolabs) were added and an extension reaction was carried out at 25 ° C for 30 minutes. The reaction product was purified by phenol / chloroform extraction and ethanol precipitation in the presence of sodium acetate. Using transcription template DNA as a template, in vitro transcription (37 ° C, 3 hours) was performed using the T7-Scribe Standard RNA IVT KIT (CELLSCRIPT) to synthesize RNA. Then, DNase (final concentration: 2U) was added, treated at 37 ° C for 30 minutes, and the gRNA was purified by phenol / chloroform extraction and ethanol precipitation. The resulting gRNA was excised using 8M Urea PAGE (8%), extracted by crushing and immersion, and purified using a 0.22 μm filter (Millipore) and gel filtration (BIO RAD). 3'ASg_GluNluc_AUI (SEQ ID NO: 12), a gRNA that edits AUA to AUI, was obtained.

[0047] Similarly, 3gTS_Nluc_IUG_F01 (SEQ ID NO: 13; FW oligo) and 3gTS_Nluc_IUG_R01 (SEQ ID NO: 14; RV oligo) were used as transcription template DNA to obtain 3gTS_Nluc_IUG (SEQ ID NO: 15), a gRNA that edits AUG to IUG.

[0048]

[0049]

[0050] (C) Annealing reaction In the combinations shown in the table below, reporter RNA (final concentration: 0.3 μM) and the corresponding gRNA (final concentration: 0.9 μM) were heated at 80°C for 3 minutes in an annealing buffer (10 mM Tri-HCl (pH 7.6), 150 mM NaCl), and then cooled to 25°C over 15 minutes to obtain an RNA complex consisting of reporter RNA and gRNA.

[0051]

[0052] (D) In ​​vitro editing reaction Assuming that the reporter RNA and gRNA were completely complexed by the annealing reaction, the following complex concentrations were calculated. The resulting RNA complex (final concentration: 0.3 μM) was dissolved in dH ２ After dilution, recombinant hADAR2 (synthesized and purified using a yeast expression system. See Macbeth, MR. and Bass, BL. Methods Enzymol. 424, 319-331 (2007), Fukuda, M. et al. Sci. Rep. srep41478 (2017)) was added to the RNA complex (final concentration: 5 nM) to a final concentration of 25 nM, and the mixture was incubated at 37 ° C. for 30 minutes.

[0053] (E) Editing Analysis After the editing reaction, reporter RNA was purified by phenol / chloroform extraction and ethanol precipitation. The resulting precipitate of each RNA complex was dissolved in 10 μL of TE buffer, and 1 μL of the solution was diluted 10-fold. Using this as a template, reverse transcription was performed using Primescript Reverse Transcriptase II (TaKaRa) and the Fluc_Sac_R01 primer (SEQ ID NO: 2) (65°C, 5 minutes, rapid cooling, 42°C, 45 minutes, 70°C, 15 minutes). The resulting cDNA was used as a template for PCR amplification (denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 20 seconds) using the pcDNA_F01 primer (SEQ ID NO: 3) and the Fluc_Sac_R01 primer (SEQ ID NO: 2) and PrimeStar GXL DNA Polymerase (TaKaRa). The amplification of the cDNA was confirmed by 0.8% agarose gel electrophoresis. The sequencing reaction (denaturation: 96°C, 10 seconds, annealing: 50°C, 5 seconds, extension: 60°C, 30 seconds) was performed using the EditCheck_Nluc R01 primer (SEQ ID NO: 4) and the SupreDye v3.1 Cycle Sequencing Kit (Edge BioSystems), followed by purification by ethanol precipitation. The resulting precipitate was dissolved in 15 μL of Hi-Di formamide (Thermo Fisher Scientific), and the base sequence was analyzed using an Applied Biosystem 3500 Genetic Analyzer (Thermo Fisher Scientific). The peak heights of A and G at the target editing site in the sequencing chromatogram obtained by the analysis were analyzed, and the editing rate (%) was calculated from the peak height ratio (G / (G+A)).

[0054] (F) In vitro translation reaction: The RNA (final: 100 ng) obtained in (D) after the editing reaction was denatured at 65 ° C for 5 minutes and then annealed by rapid cooling. 2.6 μL of the annealed sample was incubated at 30 ° C for 1 hour with 7 μL of Rabbit Reticulocyte Lysate (Promega), 0.1 μL of Amino Acid Mixture Minus Leucine, 0.1 μL of Amino Acid Mixture Minus Methionine, and 0.2 μL of RNase Inhibitor.

[0055] (G) Luciferase reporter assay Nano DLR ＴＭ Stop & Glo （Ｒ） Substrate and Nano DLR ＴＭ Stop & Glo （Ｒ） The Nluc luminescence substrate reagent was prepared by mixing 40 μL of ONE-Glo with 1:100 Buffer (Promega). Five pipetting cycles, spinning down, and setting were carried out over a one-minute period. The luminescence intensity of Fluc was measured using a GLOMAX 20 / 20 Luminometer (Promega). The measured sample was removed and analyzed with Nano-Glo. （Ｒ） 40 μL was added, and the process of pipetting five times, spinning down, and setting was carried out over one minute, and the luminescence intensity of Nluc was measured using GLOMAX.

[0056] (H) Results of editing analysis The results of the editing rates of reporter RNAs Nluc rep. AUA and Nluc rep. AUG are shown in Figure 2. As shown in Figure 2 (B), the editing rate of the target adenosine of Nluc rep. AUA was 93.2%, indicating that Nluc rep. AUI was generated by the A-to-I RNA editing reaction. Similarly, as shown in Figure 2 (C), the editing rate of the target adenosine of Nluc rep. AUG was 81.6%, indicating that Nluc rep. IUG was generated by the A-to-I RNA editing reaction.

[0057] (I) Results of translation analysis by luminescence intensity measurement The results of the reporter assay after the in vitro translation reaction of reporter RNA are shown in Figure 3. Based on the relative luminescence intensity values, when editing was induced at Nluc rep. AUA, in which the start codon of Nluc was replaced with AUA, and editing was performed to change the AUA to AUI, the relative luminescence intensity increased by approximately 4.5-fold (Figures 3(A) to 3(C)). When the relative luminescence intensities of the AUA, AUG, and AUI reporter RNAs were compared, the results were AUA < AUI < AUG. By converting AUA at the start codon position of Nluc to AUI, an increase in luminescence intensity was confirmed, indicating that AUI functions as a start codon. In other words, this suggests the possibility that a new translation region can be generated by converting the AUA sequence present in the 5'-UTR to AUI by editing.

[0058] Furthermore, as shown in Figure 4, when editing was induced at Nluc rep. AUG and editing AUG → IUG, the relative luminescence intensity decreased to about 1 / 5, and it was reduced to the same level as the luminescence intensity of Nluc rep. GUG. From this, it is thought that the conversion of AUG → IUG significantly reduces its function as a start codon (AUG ≠ IUG ≒ GUG). When editing was induced at the Nluc start codon AUG to IUG, a significant decrease in luminescence intensity was confirmed. This suggests that by converting the start codon of the uORF present in the 5'-UTR or the AUG sequence to IUG by editing, it is possible to enhance the translation amount of the downstream translation region due to the disappearance of the upstream translation start point.

[0059] Test Example 2: Evaluation of post-editing translation of model reporter RNA using two luciferases in a cell-free environment (A) Synthesis of reporter RNA (Luc rep. AUA, Luc rep. AUG) Using pcDNA3.1 Hygro(-)_Luc rep. AUA (or AUG) (SEQ ID NO: 17; XXX is ATA or ATG) as a template, template DNA was prepared by PCR (denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 20 seconds) using T7proGG (SEQ ID NO: 1) and Fluc_Sac_R01 primer (SEQ ID NO: 2) and PrimeStar GXL DNA Polymerase (TaKaRa), and purified by phenol / chloroform extraction and ethanol precipitation. RNA was synthesized by in vitro transcription (37°C, 3 hours) using the T7-Scribe Standard RNA IVT KIT (CELLSCRIPT). DNase (final concentration: 2 U) was then added, and the mixture was treated at 37°C for 30 minutes. The RNA was then purified by phenol / chloroform extraction and ethanol precipitation. The resulting RNA was purified by gel filtration (BIO RAD). The structures of the reporter RNAs (Luc rep. AUA and Luc rep. AUG) are shown in Figure 5.

[0060]

[0061] (B) gRNA synthesis In the same manner as in (B) gRNA synthesis of Test Example 1, 3'ASg_GluNluc_AUI (SEQ ID NO: 13), a gRNA that edits AUA to AUI, and 3gTS_Nluc_IUG (SEQ ID NO: 16), a gRNA that edits AUG to IUG, were obtained.

[0062] (C) Annealing reaction The procedure was carried out in the same manner as described in (C) Annealing reaction in Test Example 1. The combinations of reporter RNA and gRNA are shown in the table below.

[0063]

[0064] (D) In ​​vitro editing reaction The procedure was performed in the same manner as described in (C) Annealing reaction in Test Example 1. (E) Editing analysis The procedure was performed in the same manner as described in (E) Editing analysis in Test Example 1.

[0065] (F) In vitro translation reaction The procedure was the same as that described in Test Example 1 (F) In vitro translation reaction.

[0066] (G) Luciferase Reporter Assay The procedure was the same as that described in Test Example 1 (G) Luciferase Reporter Assay.

[0067] (H) Results of editing analysis The results of the reporter RNA Luc rep. AUA and Luc rep. AUG editing rates are shown in Figure 6. As shown in Figure 6(B), the target adenosine editing rate of Luc rep. AUA was 90.9%, indicating that Luc rep. AUI was generated by the A-to-I RNA editing reaction. Similarly, as shown in Figure 6(C), the target adenosine editing rate of Luc rep. AUG was 86.8%, indicating that Luc rep. IUG was generated by the A-to-I RNA editing reaction.

[0068] (I) Results of translation analysis by luciferase reporter assay The results of the reporter assay after the in vitro translation reaction of reporter RNA are shown in Figure 7. With Luc rep. AUA, the luminescence intensity of Nluc after editing increased by approximately 4.6-fold compared to before editing. This is thought to indicate that the AUI codon functioned as a translation initiation point, which is consistent with the results for Nluc rep. AUA shown in Figure 3. On the other hand, despite the generation of a new translation region upstream, no significant difference was observed in the luminescence intensity of the mORF Fluc before and after the editing reaction. This result suggests that the strength of the AUI triplet as an initiation codon, i.e., its binding strength with the 43S preinitiation complex (PIC), is weaker than that of AUG. With Luc rep. AUG, the luminescence intensity of Nluc after editing decreased to approximately 1 / 12 compared to before editing, and was reduced to the same level as the luminescence intensity of Luc rep. GUG. This is thought to have been converted to IUG and thus no longer function as a translation initiation site. Furthermore, in contrast to Luc rep. AUA, the luminescence intensity of the mORF Fluc increased approximately 1.7-fold upon deletion of the upstream translation region (Figure 4). This result suggests that if the presence of the uORF potentially represses mORF translation, the repressive effect can be avoided by converting the uORF start codon (uAUG) to an IUG triplet through RNA editing.

[0069] Test Example 3: Evaluation of translational repression function of each uAUG in endogenous 5'-UTR in cell (A) Cell culture: Human embryonic kidney cells (HEK293 cells), in which ADAR2 expression can be induced by the Tet-on system, were cultured in a medium containing Dulbecco's Modified Eagle's Medium-high glucose (SIGMA), 10% Tet System Approved FBS (TaKaRa), 0.025% Trypsin-EDTA Solution (SIGMA), 1 μg / mL puromycin, and 100 μg / mL G418 at 37°C and 6.5% CO. ２ When the 10 cm culture dish reached 80% confluency, the cells were cultured at 1.0 × 10 ５The cells were subcultured at 1000 cells / well onto a 24-well plate and cultured for 48 hours with Dox (5.0 μg / mL) to induce ADAR2 expression. After 48 hours, the cells were transfected with 10 ng of pcDNA3.1 Hygro(-)_PKIG rep. series (SEQ ID NO: 18) using Lipofectamine 3000 Transfection Kit (Invitrogen) and cultured for an additional 48 hours.

[0070] The PKIG (cAMP-Dependent Protein Kinase Inhibitor Gamma) gene used here was selected by searching the A-to-I RNA editing database RADAR to find genes with 5'-UTRs that can generate or delete translation initiation sites through RNA editing. The base sequence of the reporter RNA template, pcDNA3.1 Hygro(-)_PKIG rep. series (SEQ ID NO: 17), is shown in the table below. A schematic diagram of the reporter RNA structure is also shown in Figure 8. As shown in Figure 8, there are five upstream initiation codons (uAUG) within the 5'-UTR of PKIG.

[0071] (B) Luciferase reporter assay of cultured cell solution After culturing as described in Test Example 3(A), the medium was aspirated using an aspirator, and 100 μL of 1× Passive Lysis Buffer (Promega) was added per well. The cells were then shaken for 15 minutes to detach the cells. The cells were then disrupted by pipetting and completely lysed. The collected cell extract was centrifuged at 4°C, 15,000 rpm, for 5 minutes, and 10 μL of the supernatant was dispensed.

[0072] Nano DLR ＴＭ Stop & Glo （Ｒ） Substrate and Nano DLR ＴＭ Stop & Glo （Ｒ） Buffer (Promega) was mixed at a ratio of 1:100 to prepare an Nluc luminescent substrate reagent.

[0073] 40 μL of ONE-Glo was added to 10 μL of cell extract, and the process of pipetting five times, spinning down, and setting down was carried out in 1 minute. The luminescence intensity of Fluc was measured using a GLOMAX 20 / 20 Luminometer (Promega). （Ｒ） 40 μL was added, and the process of pipetting five times, spinning down, and setting was carried out over one minute, and the luminescence intensity of Nluc was measured using GLOMAX.

[0074] (C) Results of translation analysis by luciferase reporter assay. Figure 9 shows the results of evaluating the translation repression function of each uAUG in the endogenous 5'-UTR. When all uAUGs in the 5'-UTR were replaced with GUGs, the mORF translation rate increased approximately 3.5-fold, indicating that uAUGs strongly repressed downstream translation. Furthermore, when the relative luminescence intensities of the wt and editing site AUGs, and AUG1 to AUG5 were compared, the wt and editing site AUGs, AUG1, and AUG2 were almost identical. This indicates that the downstream repression effect is not cooperatively suppressed by multiple AUGs, but is largely suppressed by a single AUG.

[0075]

[0076] Test Example 4: Evaluation of post-editing translation of reporter RNA based on endogenous 5'-UTR in a cell-free environment (A) Synthesis of reporter RNA Using pcDNA3.1 Hygro(-)_PKIG rep. AUA_(GUG) or AUG_(GUG) (SEQ ID NO: 18; XXXX = AATA or AATG) as a template, T7proGG (SEQ ID NO: 1) and Fluc_Sac_R01 primer (SEQ ID NO: 2), and PrimeStar GXL DNA Polymerase (TaKaRa), PCR was performed (denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 20 seconds) to prepare template DNA. The template DNA was then extracted with phenol / chloroform and purified by ethanol precipitation. Reporter RNA was synthesized by in vitro transcription (37°C, 3 hours) using the T7-Scribe Standard RNA IVT KIT (CELLSCRIPT). DNase (final concentration: 2 U) was then added, and the mixture was treated at 37°C for 30 minutes. The RNA was then purified by phenol / chloroform extraction and ethanol precipitation. The resulting RNA was purified by gel filtration (BIO RAD).

[0077] These reporter RNAs were prepared by mutating all uAUG sequences in the 5'-UTR of the PKIG gene used in Test Example 3 to GUG, and changing the portion shown as the editing site in Figure 8 to AUA (wild type) or AUG.

[0078]

[0079] (B) gRNA synthesis The operation was carried out in the same manner as described in (B) gRNA synthesis in Test Example 1. However, the oligo DNA (primer) described below was used to obtain 3'ASg_GluPKIG_AUI (SEQ ID NO: 21), a gRNA that edits AUA to AUI, and 5'ASg_GluPKIG_UIUG (SEQ ID NO: 24), a gRNA that edits AUG to IUG.

[0080]

[0081] (D) In ​​vitro editing reaction The procedure was carried out in the same manner as described in (C) Annealing reaction in Test Example 1. The combinations of reporter RNA and gRNA are shown in the table below.

[0082]

[0083] (E) Editing Analysis The procedure was carried out in the same manner as described in (E) Editing Analysis in Test Example 1.

[0084] (F) In vitro translation reaction The procedure was the same as that described in Test Example 1 (F) In vitro translation reaction.

[0085] (G) Luciferase Reporter Assay The procedure was the same as that described in Test Example 1 (G) Luciferase Reporter Assay.

[0086] (H) Results of editing analysis The results of the reporter RNA PKIG rep. AUA_(GUG) and PKIG rep. AUG_(GUG) editing rates are shown in Figure 9. The target adenosine editing rate of PKIG rep. AUA_(GUG) was 92.2%, indicating that Luc rep. AUI was generated by the A-to-I RNA editing reaction. Similarly, the target adenosine editing rate of PKIG rep. AUG_(GUG) was 84.3%, indicating that Luc rep. IUG was generated by the A-to-I RNA editing reaction.

[0087] (I) Results of translation analysis by luciferase reporter assay The results of the reporter assay after the in vitro translation reaction of reporter RNA are shown in Figures 11 and 12. As shown in Figure 11, when the PKIG 5'-UTR was converted from AUA to AUI, no significant difference was observed in the relative luminescence intensity before and after the editing reaction, while a slight decrease in the average value was observed. As shown in Figure 12, in the AUG → IUG conversion, the relative luminescence intensity after editing increased by approximately 2.2 times compared to before editing, indicating enhanced translation of the downstream Main ORF due to the disappearance of the putative uORF in the 5'-UTR. This result is consistent with the increase in luminescence intensity of Fluc in Luc rep. AUG in Test Example 3. The above indicates that protein translation can be controlled through the deletion of the translation start point in the 5'-UTR (AUG → IUG).

[0088] Test Example 5: Induction of editing into PKIG rep. AUA_(GUG) and PKIG rep. AUG_(GUG) using modified nucleic acids in cultured cells and evaluation of translation (A) Cell culture: HEK293 cells (JCRB cell bank) were cultured in a 24-well plate at 1.0 × 10 ５ The cells were seeded at 1000 cells / well. After 48 hours, the reporter plasmid (final concentration 10 ng), hADAR2 plasmid (final concentration 500 ng), and chemically modified RNA-edited nucleic acid (final concentration 50 nM) were transfected using Lipofectamine 3000 Transfection Kit (Invitrogen). After 48 hours, the cells were harvested.

[0089] The reporter plasmid used was pcDNA3.1 Hygro(-)_PKIG rep. AUA_(GUG) (SEQ ID NO: 18; XXXX = AATA), or pcDNA3.1 Hygro(-)_PKIG rep. AUG_(GUG) (SEQ ID NO: 18; XXXX = UATG). The ADAR2 plasmid used was pcDNA3.1 Hygro(-)_hADAR2_E488Q (SEQ ID NO: 27). The chemically modified RNA-editing nucleic acids used were AD2gPKIG_AUA (SEQ ID NO: 25) and AD2gPKIG_UAUG (SEQ ID NO: 26), which are modified gRNAs shown in the table below. In the table below, capital letters represent ribonucleosides, lowercase letters represent deoxynucleosides, and underlines represent 2'-O-methylribonucleosides. In the modified gRNA, each nucleoside is linked by a phosphorothioate bond.

[0090]

[0091]

[0092] (B) Editing analysis. Total RNA was extracted from cells cultured in a 24-well plate using Sepasol RNA I Super G (Nacalai Tesque). The resulting total RNA was treated with 10 U of DNase I and purified by ethanol precipitation. Using 0.5 μg of the resulting total RNA as a template, reverse transcription was performed using PrimeScript II Reverse Transcriptase (TaKaRa) and Oligo(dT)RV primer (SEQ ID NO: 8), and cDNA was amplified. Fragments derived from reporter RNA were amplified by nested PCR. First, a first PCR (denaturation: 98°C, 10 seconds, annealing: 55°C, 15 seconds, extension: 68°C, 20 seconds) was performed using PrimeStar GXL DNA polymerase (TaKaRa), T7proGG primer (SEQ ID NO: 4), and 3'-adp primer (SEQ ID NO: 9). Using the cDNA obtained by diluting the first PCR product 400-fold as a template, a second PCR (denaturation: 98°C, 10 seconds, annealing: 55°C, 15 seconds, extension: 68°C, 20 seconds) was performed using PrimeStar GXL DNA polymerase (TaKaRa), pcDNA_F01 primer (SEQ ID NO: 6), and Fluc_Sac_R01 primer (SEQ ID NO: 5) to amplify the fragment. Amplification was confirmed by 0.8% agarose gel electrophoresis. The sequencing reaction (denaturation: 96°C, 10 seconds, annealing: 50°C, 5 seconds, extension: 60°C, 30 seconds) was performed using the EditCheck_Nluc_R01 primer (SEQ ID NO: 7) and the SupreDye v3.1 Cycle Sequencing Kit (Edge BioSystems), followed by purification by ethanol precipitation. The resulting precipitate was dissolved in 15 μL of Hi-Di formamide (Thermo Fisher Scientific), and the base sequence was analyzed using an Applied Biosystem 3500 Genetic Analyzer (Thermo Fisher Scientific). The peak heights of A and G at the target editing site in the resulting sequencing chromatogram were analyzed, and the editing rate (%) was calculated from the peak height ratio (G / (G+A)).

[0093] (C) Luciferase reporter assay of cultured cell solution The procedure was the same as that described in Test Example 3 (B) Luciferase reporter assay of cultured cell solution.

[0094] (D) Results of editing analysis The results of editing induction in cultured cells are shown in Figure 13. The editing rate of the target adenosine of PKIG rep. AUA_(GUG) was 77.3%, and the editing rate of the target adenosine of KIG rep. AUG_(GUG) was 31.8%, respectively.

[0095] (E) Results of translation analysis by luciferase reporter assay The results of measuring the luminescence intensity of cell extracts under the above-mentioned editing efficiency environment are shown in Figures 14 and 15. When AUA → AUI conversion of the cellular 5'-UTR was performed in cultured cells, a tendency for the relative luminescence intensity to decrease after editing compared to before editing was observed. In the AUG → IUG conversion, enhanced translation of the downstream mORF was observed due to the disappearance of the putative uORF in the 5'-UTR. This result is consistent with the results of the reporter assay after the editing reaction of Luc rep. AUG and the results of the reporter assay after the in vitro editing reaction of PKIG rep. AUG_(GUG) (Test Example 4). In summary, we demonstrate that A-to-I RNA editing in the 5'-UTR can control the amount of protein translation by generating an additional uORF (AUA → AUI conversion) and deleting the translation start site of the uORF (AUG → IUG conversion), and model experiments demonstrate that these regulatory mechanisms exist in cells.

[0096] Test Example 6: Searching for genes capable of translational control by A-to-I RNA editing of 5'-UTR using a database (A) Obtaining human endogenous 5'-UTR sequences All registered human endogenous 5'-UTR sequences classified under protein_coding were obtained using the genome sequence database Ensembl Biomart.

[0097] (B) Extraction of genes whose translation rate can be controlled by A-to-I RNA editing. The sequences obtained in (A) were classified according to the presence or absence of an AUG sequence. Then, genes whose translation rate can be controlled by A-to-I RNA editing (1) AUA → AUI (reducing translation rate) or (2) AUG → IUG (increasing translation rate) were extracted by the following procedure.

[0098] (1) Among genes that do not have an AUG sequence in the 5'-UTR, those with one or more AUA sequences were extracted as candidates for AUA → AUI control. A total of 7,525 genes were found, with the number of AUA sequences ranging from 1 to 8.

[0099] (2) Genes with an AUG sequence in the 5'-UTR were extracted as candidates for AUG → IUG regulation, and the number of gene hits was 17,009.

[0100] Test Example 7: Evaluation of translational regulation via A-to-I RNA editing of the 5'-UTR of genes searched from a database

[0101] (A) From the genes searched for in Test Example 6, (1) the UGP2 gene was selected from the group of genes capable of AUA → AUI regulation (repression of translation amount), and (2) the ASF1A gene was selected from the group of genes capable of AUG → IUG regulation (enhancement of translation amount), and the respective 5'-UTR sequences were used to synthesize reporter plasmids pcDNA3.1 Hygro(-)_UGP2 rep. AUA (SEQ ID NO: 28) and pcDNA3.1 Hygro(-)_ASF1A rep. AUG (SEQ ID NO: 29). The structures of reporter RNAs expressed upon transfection of these plasmids into cells are shown as schematic diagrams in Figures 16 and 17.

[0102] (B) Cell culture: HEK293 cells (JCRB Cell Bank) were cultured in a 24-well plate at 1.0 × 10 ５The cells were seeded at 1000 cells / well. After 48 hours, the reporter plasmid (final concentration 10 ng), hADAR2 plasmid (final concentration 500 ng), and chemically modified RNA-edited nucleic acid (final concentration 50 nM) were transfected using Lipofectamine 3000 Transfection Kit (Invitrogen). After 48 hours, the cells were harvested.

[0103] The reporter plasmid used was pcDNA3.1 Hygro(-)_UGP2 rep. AUA (SEQ ID NO: 28) or pcDNA3.1 Hygro(-)_ASF1A rep. AUG (SEQ ID NO: 29). The ADAR2 plasmid used was pcDNA3.1 Hygro(-)_hADAR2_E488Q (SEQ ID NO: 27). The chemically modified RNA-editing nucleic acids used were the modified gRNAs AD2gUGP2_AUA (SEQ ID NO: 30) and AD2gASF1A_AUG (SEQ ID NO: 31) shown in the table below. In the table below, capital letters represent ribonucleosides, lowercase letters represent deoxynucleosides, and underlines represent 2'-O-methylribonucleosides. In the modified gRNA, each nucleoside is linked by a phosphorothioate bond.

[0104]

[0105]

[0106]

[0107] (C) Editing analysis. Total RNA was extracted from cells cultured in a 24-well plate using Sepasol RNA I Super G (Nacalai Tesque). The resulting total RNA was treated with 10 U of DNase I and purified by ethanol precipitation. Using 0.5 μg of the resulting total RNA as a template, reverse transcription was performed using PrimeScript II Reverse Transcriptase (TaKaRa) and Oligo(dT)RV primer (SEQ ID NO: 8), and cDNA was amplified. Fragments derived from reporter RNA were amplified by nested PCR. First, a first PCR (denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 20 seconds) was performed using PrimeStar GXL DNA polymerase (TaKaRa), T7proGG primer (SEQ ID NO: 4), and 3'-adp primer (SEQ ID NO: 9). Using the cDNA obtained by 400-fold dilution of the first PCR product as a template, second PCR (denaturation: 98°C, 10 seconds; annealing: 55°C, 15 seconds; extension: 68°C, 20 seconds) was performed using PrimeStar GXL DNA polymerase (TaKaRa), UGP2_Nhe_F01 primer (SEQ ID NO: 32) or ASF1A_Nhe_F01 primer (SEQ ID NO: 33), and Fluc_Sac_R01 primer (SEQ ID NO: 5) to amplify the fragment. Amplification was confirmed by 0.8% agarose gel electrophoresis. The sequencing reaction (denaturation: 96°C, 10 seconds, annealing: 50°C, 5 seconds, extension: 60°C, 30 seconds) was performed using the EditCheck_Nluc_R01 primer (SEQ ID NO: 7) and the SupreDye v3.1 Cycle Sequencing Kit (Edge BioSystems), followed by purification by ethanol precipitation. The resulting precipitate was dissolved in 15 μL of Hi-Di formamide (Thermo Fisher Scientific), and the base sequence was analyzed using an Applied Biosystem 3500 Genetic Analyzer (Thermo Fisher Scientific).The peak heights of A and G at the target editing site in the resulting sequencing chromatogram were analyzed, and the editing rate (%) was calculated from the peak height ratio (G / (G+A)).

[0108]

[0109] (D) Luciferase reporter assay of cultured cell solution The procedure was the same as that described in Test Example 3 (B) Luciferase reporter assay of cultured cell solution.

[0110] (E) Results of editing analysis The results of editing induction in cultured cells are shown in Figure 18. The editing rate of the target adenosine for UGP2 rep. AUA was 68.6%, and the editing rate of the target adenosine for ASF1A rep. AUG was 28.2%, respectively.

[0111] (F) Results of translational control analysis by luciferase reporter assay using the 5'-UTR of genes searched from the database. The results of measuring the luminescence intensity of cell extracts under the above-mentioned editing efficiency environment are shown in Figures 19 and 20. Even when the natural 5'-UTR expressed in the cells was converted from AUA to AUI, a tendency for the relative luminescence intensity to decrease after editing compared to before editing was observed. The conversion from AUG to IUG showed enhanced translation of the downstream mORF due to the disappearance of the predicted uORF in the 5'-UTR. These results are consistent with the results of the intracellular editing experiments using the model sequence reporters PKIG rep. AUA_(GUG) and PKIG rep. UAUG_(GUG) in Test Example 5. In summary, we demonstrated that A-to-I RNA editing can control the amount of protein translation in the native 5'-UTRs obtained from the database by generating additional uORFs (AUA → AUI conversion) and deleting the translation start site of the uORF (AUG → IUG conversion).

[0112] The disclosure of Japanese Patent Application No. 2024-101511 (filing date: June 24, 2024) is incorporated herein by reference in its entirety. All documents, patent applications, and technical standards mentioned herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A method for controlling the translation amount of a target protein, comprising editing a base sequence represented by AUA or AUG contained in the 5' untranslated region of mRNA containing the coding region of the target protein into a base sequence represented by AUI or IUG in the presence of adenosine deaminase.

2. A method for controlling the translation amount of a target protein, comprising contacting, in the presence of adenosine deaminase, mRNA comprising a 5' untranslated region containing a base sequence represented by AUA or AUG and a coding region of a target protein with a target editing guide oligonucleotide capable of editing the base sequence represented by AUA or AUG contained in the 5' untranslated region into a base sequence represented by AUI or IUG in the presence of adenosine deaminase.

3. The method according to claim 2, which comprises editing at least one of the base sequences represented by AUG contained in the 5' untranslated region to a base sequence represented by IUG.

4. The method according to claim 3, wherein the number of base sequences represented by AUG contained in the 5' untranslated region after editing is set to 1 or less.

5. The method according to claim 2, which comprises editing at least one of the base sequences represented by AUA contained in the 5' untranslated region into a base sequence represented by AUI.

6. The method according to claim 5, wherein the number of base sequences represented by AUG contained in the 5' untranslated region is 0.

7. The method according to any one of claims 2 to 6, wherein the 5' untranslated region contains from 6 to 1200 ribonucleotide residues.

8. The method according to any one of claims 2 to 6, wherein the base sequence represented by AUA or AUG is present at a position between 4 and 1200 residues from the 5' capping of the mRNA.

9. The method of any one of claims 2 to 8, wherein the target editing guide oligonucleotide comprises a first oligonucleotide that forms a complementary strand with the mRNA and a second oligonucleotide linked to the 5' side of the first oligonucleotide, wherein the first oligonucleotide comprises a target-corresponding nucleotide residue corresponding to the third adenosine residue from the 5' side of a base sequence represented by AUA in the 5' untranslated region of the mRNA, or an adenosine residue in a base sequence represented by AUG, an oligonucleotide of 10 to 24 residues linked to the 3' side of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, and an oligonucleotide of 3 to 6 residues linked to the 5' side of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, and the second oligonucleotide comprises an oligonucleotide of 8 to 50 residues and has a base sequence capable of forming a stem-loop structure within the molecule.

10. The method of any one of claims 2 to 8, wherein the target editing guide oligonucleotide comprises a third oligonucleotide that forms a complementary strand with the mRNA and a fourth oligonucleotide linked to the 3' side of the third oligonucleotide, wherein the third oligonucleotide comprises a target-corresponding nucleotide residue corresponding to the third adenosine residue from the 3' side of a base sequence represented by AUA in the 5' untranslated region of the mRNA, or an adenosine residue in a base sequence represented by AUG, an oligonucleotide of 10 to 24 residues linked to the 5' side of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, and an oligonucleotide of 3 to 6 residues linked to the 3' side of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, and the fourth oligonucleotide comprises an oligonucleotide of 8 to 50 residues and has a base sequence capable of forming a stem-loop structure within the molecule.

11. The method according to any one of claims 2 to 8, wherein the target editing guide oligonucleotide comprises a fifth oligonucleotide that forms a complementary strand with the mRNA and a sixth oligonucleotide linked to the 5' side of the fifth oligonucleotide, wherein the fifth oligonucleotide comprises a target-corresponding nucleotide residue corresponding to the third adenosine residue from the 5' side of a base sequence represented by AUA in the 5' untranslated region of the mRNA, or an adenosine residue in a base sequence represented by AUG, an oligonucleotide of 10 to 24 residues linked to the 3' side of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, and an oligonucleotide of 3 to 6 residues linked to the 5' side of the target-corresponding nucleotide residue and having a base sequence complementary to the mRNA, wherein the sixth oligonucleotide lacks a nucleotide residue corresponding to a nucleotide residue in the mRNA at its 3' end or has a nucleotide residue that does not form a complementary pair with a nucleotide residue in the mRNA, and wherein the sixth oligonucleotide has 2 to 10 residues, and at least nucleotide residues other than those at the 3' end form a double-stranded structure complementary to the mRNA.

12. A method for controlling the translation amount of a target protein, comprising editing a base sequence represented by AUG, which is the start codon of mRNA containing the coding region of the target protein, into a base sequence represented by IUG in the presence of adenosine deaminase.

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