WO2024161623A1 - Readthrough drug, use of nucleic acid probe and readthrough combination agent - Google Patents

Abstract

This readthrough drug contains a nucleic acid probe. The nucleic acid probe comprises a base sequence complementary to an mRNA targeted for modification and a reactive base derivative at a position complementary to an adenine contained in a stop codon as a target for modification in mRNA wherein the transfer group contained in the reactive base derivative is transferred to the amino group at position six of adenine.

Description

Use of read-through drugs, read-through combinations and nucleic acid probes

The present invention relates to the use of lead-through drugs, lead-through combinations, and nucleic acid probes.

A nonsense mutation is a mutation that changes a codon that codes for an amino acid into a stop codon. The stop codon resulting from the mutation is called a premature termination codon (PTC). Nonsense mutations account for approximately 20% of point mutations that affect the coding region of genes. Stop codons occurring in coding regions can lead to exon skipping, mRNA degradation by the nonsense-mediated mRNA decay (NMD) mechanism, and the production of short, nonfunctional polypeptide chains. The impact of PTC on the body is serious, and it can cause many intractable diseases, including genetic disorders such as Dischenne muscular dystrophy (DMD) and cystic fibrosis (CF), as well as cancer.

There is a phenomenon called read-through, in which the action of certain compounds causes the PTC to be skipped and translation to continue. Compounds that promote read-through are expected to synthesize proteins similar to the wild-type normal protein, improving nonsense mutation genetic diseases and treating the disease.

Patent Document 1 discloses aminoglycoside compounds as compounds with read-through activity. Furthermore, Patent Document 2 shows that aminoglycoside compounds such as G418 have read-through activity against PTC in the COL7A1 gene, which causes dystrophic epidermolysis bullosa. Patent Document 3 also discloses a method of inducing an immune response using ataluren as a compound that promotes read-through.

Special Publication No. 2009-532461 JP 2016-222681 A Special table 2019-505578 publication

The fundamental problem with the compounds with the above-mentioned read-through activity is that they cannot distinguish between PTCs and normal stop codons. In many cases, they read through not only the targeted PTC but also normal stop codons, adding extra peptides to normal proteins, raising concerns about side effects from the added peptides.

On the other hand, genome editing is expected to be a treatment for hereditary diseases caused by PTC. Genome editing is a technique that involves site-specific cleavage of the double strand of genomic DNA, followed by repairing the DNA by non-homologous end joining or homologous recombination to induce mutations. However, at present, although genome editing is a method suitable for ex vivo or fertilized eggs, it is difficult to apply it to the patient's entire body.

The present invention has been made in consideration of the above circumstances, and aims to provide a read-through drug that has sequence-selective read-through activity against stop codons, a combination drug for read-through, and the use of a nucleic acid probe for producing the read-through drug.

The lead-through drug according to the first aspect of the present invention comprises:
Contains a nucleic acid probe,
The nucleic acid probe comprises:
a base sequence complementary to an mRNA to be modified, and a reactive base derivative at a position complementary to an adenine contained in a termination codon to be targeted for modification in the mRNA;
The transfer group contained in the reactive base derivative is transferred to the 6-amino group of the adenine.

The reactive base derivative is
As shown in formula I:
This may also be the case.

(In formula I, R is H or a substituent that includes a linker terminated with an alkynyl group.)

R is a substituent containing a linker having an alkynyl group at its terminal end,
an atomic group is added to the substituent via the alkynyl group in the transfer group transferred to the mRNA;
This may also be the case.

As the atomic group, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, glucose, G418, amikacin, doxorubicin or negamycin is added to the substituent via a hydroxy group or an amino group;
This may also be the case.

The adenine contained in the stop codon is
A at the end of UAA, A at the end of UAG, or A at the end of UGA,
The linker is
A triethylene glycol linker or a tetraethylene glycol linker,
As the atomic group, α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin is added to the substituent via a hydroxy group;
This may also be the case.

The adenine contained in the stop codon is
The terminal A of UAA,
As the atomic group, doxorubicin or negamycin is added to the substituent via an amino group;
This may also be the case.

The lead-through combination according to the second aspect of the present invention comprises:
A lead-through drug according to the first aspect of the present invention;
a compound having the atomic group and donating the atomic group to the substituent;
Equipped with.

The use of the nucleic acid probe according to the third aspect of the present invention comprises:
1. Use of a nucleic acid probe for the manufacture of a read-through drug, comprising:
The nucleic acid probe comprises:
a base sequence complementary to an mRNA to be modified, and a reactive base derivative at a position complementary to an adenine contained in a termination codon to be targeted for modification in the mRNA;
The transfer group contained in the reactive base derivative is transferred to the 6-amino group of the adenine.

According to the present invention, read-through activity can be obtained for stop codons in a sequence-selective manner.

FIG. 1 is a diagram illustrating the synthesis of a nucleic acid probe, the synthesis of a chemically modified mRNA by using the nucleic acid probe, and the addition of a compound to the chemically modified mRNA by a click reaction. FIG. 1 shows structures of azide derivatives. FIG. 2 shows the base sequence of mRNA used in the Examples and the amino acid sequence of the peptide translation product thereof. FIG. 1 shows an example of an HPLC chromatogram tracking the process of synthesis of a nucleic acid probe and production of chemically modified mRNA. A shows the peak of the probe. B shows the peak of the probe into which a transfer group has been introduced. C shows the peak of the mRNA into which a transfer group has been transferred. D shows the peak of the chemically modified mRNA obtained by a click reaction. E shows the peak of a sample isolated by high performance liquid chromatography (HPLC). This is a diagram showing a total ion current (TIC) chromatogram obtained by analyzing a translation reaction product of an mRNA containing the codon CAA. A shows the peak of the translation reaction product of an mRNA before chemical modification. B shows the peak of the translation reaction product of an mRNA in which a transfer group having pyrene has been introduced via a triethylene glycol linker (3EG) to the C of the codon CAA. C shows the peak of the translation reaction product of an mRNA in which a transfer group having β-cyclodextrin (CD) has been introduced via 3EG to the A at the terminal of the codon CAA. D shows the peak of the translation reaction product of an mRNA in which a transfer group having pyrene has been introduced via 3EG to the A at the terminal of the codon CAA. This is a diagram showing TIC chromatograms analyzing the translation reaction product of mRNA into which a transfer group has been introduced at the terminal A of the codon UAA. A shows the peak of the translation reaction product of mRNA before chemical modification. B shows the peak of the translation reaction product of mRNA into which a pyridinylketovinyl transfer group has been introduced. C shows the peak of the translation reaction product of mRNA into which a transfer group having 3EG has been introduced. D shows the peak of the translation reaction product of mRNA into which a transfer group having α-CD has been introduced via 3EG. E shows the peak of the translation reaction product of mRNA into which a transfer group having β-CD has been introduced via 3EG. F shows the peak of the translation reaction product of mRNA into which a transfer group having γ-CD has been introduced via 3EG. FIG. 1 shows the results of investigating the effect of the length of the ethylene glycol linker on mRNA containing the codon CAA into which a transfer group having a CD has been introduced via an ethylene glycol linker. This is a TIC chromatogram of the translation reaction product of mRNA in which a transfer group has been introduced to the A at the end of the codon UAA. A shows the peak of the translation reaction product of mRNA in which a transfer group having glucose has been introduced via a diethylene glycol linker (2EG). B shows the peak of the translation reaction product of mRNA in which a transfer group having glucose has been introduced via a 3EG. C shows the peak of the translation reaction product of mRNA in which a transfer group having glucose has been introduced via a tetraethylene glycol linker (4EG). D shows the peak of the translation reaction product of mRNA in which a transfer group having G418 has been introduced via 3EG. E shows the peak of the translation reaction product of mRNA in which a transfer group having amikacin has been introduced via 3EG. F shows the peak of the translation reaction product of mRNA in which a transfer group having doxorubicin has been introduced via 3EG. G shows the peak of the translation reaction product of mRNA in which a transfer group having negamycin has been introduced via 3EG. This is a diagram showing TIC chromatograms obtained by analyzing the translation reaction products of mRNA in which a transfer group has been introduced into the A of the codon UAG or the A of the codon UGA. A shows the peak of the translation reaction product of mRNA containing the codon UAG before chemical modification. B shows the peak of the translation reaction product of mRNA in which a pyridinylketovinyl transfer group has been introduced into the A of the codon UAG. C shows the peak of the translation reaction product of mRNA in which a transfer group having β-CD has been introduced into the A of the codon UAG via 3EG. D shows the peak of the translation reaction product of mRNA containing the codon UGA before chemical modification. E shows the peak of the translation reaction product of mRNA in which a pyridinylketovinyl transfer group has been introduced into the A of the codon UGA. F shows the peak of the translation reaction product of mRNA in which a transfer group having β-CD has been introduced into the A of the codon UGA via 3EG. FIG. 1 shows the relationship between the production rate of translation reaction products of unmodified mRNA and translation reaction products of mRNA into which a pyridinylketovinyl transfer group has been introduced, and the concentration of peptide release factor (RF2).

The following describes an embodiment of the present invention with reference to the drawings. Note that the present invention is not limited to the following embodiment and drawings. Note that in the following embodiment, the expressions "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of".

The read-through drug according to this embodiment includes a nucleic acid probe. The nucleic acid probe is an oligonucleotide having a base sequence complementary to the mRNA to be modified. The nucleic acid probe is preferably RNA. The base length of the nucleic acid probe is not particularly limited, but is, for example, 8 to 30 bases, 9 to 25 bases, or 10 to 20 bases. The base length of the nucleic acid probe is preferably 11 bases.

The mRNA to be modified is an mRNA having a stop codon, particularly a PTC that causes a disease. Such diseases include, for example, DMD, Becker muscular dystrophy (BMD), CF, and cancer. For example, the mRNA to be modified can be determined based on a known PTC that causes a disease. The location of the PTC in the dystrophin gene that causes DMD and BMD is shown in Table 1. For example, a DNA mutation in Table 1, c. 354G>A, means that the 354th base G counting from the 5' end of the dystrophin gene is mutated to A, resulting in a PTC. For example, a protein mutation in Table 1, p. Trp118X, means that the codon corresponding to tryptophan, the 118th amino acid residue counting from the N-terminus of the dystrophin protein, is a PTC.

With regard to cancer, the position of the PTC in the p53 gene and the base sequence containing the PTC are exemplified in Table 2. For example, W53X in the protein mutation in Table 2 means that the codon corresponding to tryptophan, the 53rd amino acid residue counting from the N-terminus of the p53 protein, is a PTC. The underlined codons in the base sequence shown in Table 2 are stop codons that are targets for modification in mRNA.

Regarding CF, the location of the PTC in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that causes CF and the base sequence including the PTC are shown in Table 3. The notation of protein mutations in Table 3 is the same as in Table 2. The underlined codons in the base sequence shown in Table 3 are stop codons that are targets for modification in mRNA.

The nucleic acid probe has a reactive base derivative at a position complementary to the adenine contained in the stop codon targeted for modification in the mRNA. For example, the reactive base derivative is shown in formula I. In formula I, R is H or a substituent containing a linker having an alkynyl group at its end.

Examples of linkers include organic molecules, peptide chains, and polyethylene glycol. The peptide chain may be one amino acid or multiple amino acids, for example, 3 to 20 amino acids. There are no particular limitations on the alkynyl group, so long as it can be used in a click reaction with azide, etc.

The nucleic acid probe transfers the transfer group contained in the reactive base derivative to the amino group at the 6-position of adenine. In the case of the reactive base derivative shown in formula I above, the transfer group is transferred to the adenine in the complementary position as follows:

When R is the above-mentioned substituent, the adenine of the mRNA can be modified through the alkynyl group of the transfer group transferred from the nucleic acid probe to the mRNA. That is, an atomic group is added to the substituent through the alkynyl group in the transfer group transferred to the mRNA. The atomic group may be a molecule having read-through activity or a molecule having no read-through activity. By azidizing such a molecule, it can be added to the substituent by a click reaction with the alkynyl group in the transfer group. During azidization, the molecule may be bonded to _N3 via a linker. The linker is arbitrary, and may be, for example, an alkyl group, a carbonyl, an amide, a disulfide, a thioether, a hydrazone, a hydrazide, an imine, an oxime, a urea, a thiourea, an amidine, an amine, a sulfonamide, or a combination thereof.

The above molecules include, for example, α-CD, β-CD, γ-CD, glucose, G418, amikacin, doxorubicin, and negamycin. These molecules are added to the substituent as an atomic group via the hydroxyl or amino group that the molecule possesses.

Preferably, when the adenine contained in the stop codon is the A at the end of UAA, the A at UAG, or the A at the end of UGA, the linker is 3EG or 4EG, and α-CD, β-CD, or γ-CD is added as the atomic group to the substituent via the hydroxy group of the molecule. Also, when the adenine contained in the stop codon is the A at the end of UAA, doxorubicin or negamycin may be added as the atomic group to the substituent via the amino group of the molecule.

The nucleic acid probe can be synthesized by a known method. For example, first, an oligonucleotide consisting of ribonucleotides having a base sequence complementary to the mRNA to be modified and having 4-thio-2'-deoxythymidine (SdT) as a nucleic acid pairing with the target adenine is synthesized using an automatic nucleic acid synthesizer or the like. Then, the oligonucleotide is reacted with (E)-3-iodo-1-(pyridin-2-yl)prop-2-en-1-one in a buffer solution. The pH of the buffer solution is preferably 9 to 11, more preferably 10. An example of the buffer solution is a carbonate buffer.

The nucleic acid probe may contain modified nucleosides constituting the nucleic acid probe. For example, at least one of the nucleosides at the 5' end and the 3' end of the nucleic acid probe may be a 2'-O-methylribonucleoside (2'-OMe).

The nucleic acid probe also includes pharmacologically acceptable salts. The salt may be either an acid salt or a basic salt. Examples of salts include alkali metal salts such as lithium salt, sodium salt, and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, oxalate, and phosphate, as well as acetate, propionate, hexanoate, cyclopentanepropionate, glycolate, pyruvate, lactate, malonate, succinate, malate, fumarate, tartrate, citrate, benzoate, o-(4-hydroxybenzoyl)benzoate, cinnamate, mandelate, methanesulfonate, and the like. Examples of organic acid salts include salts of ethanesulfonate, 1,2-ethanedisulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-chlorobenzenesulfonate, 2-naphthalenesulfonate, p-toluenesulfonate, camphorsulfonate, glucoheptanoate, 3-phenylpropionate, trimethyl acetate, tert-butyl acetate, lauryl sulfate, gluconate, glutamate, hydroxynaphthoate, salicylate, stearate, trifluoroacetate (TFA) salt, maleate, and muconate.

The lead-through drug according to this embodiment may contain any pharmacologically acceptable component in addition to the nucleic acid probe. The optional components include, for example, carriers, excipients, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, isotonicity agents, buffers, and soothing agents. In addition, additives such as preservatives, antioxidants, colorants, and sweeteners may be added to the lead-through drug as necessary.

The administration route of the lead-through drug in this embodiment is not particularly limited. The lead-through drug may be administered, for example, parenterally or orally. In the case of parenteral administration, it may be intravenous injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, transdermal administration, nasal administration, pulmonary administration, enteral administration, transmucosal administration, etc. The lead-through drug may be administered via infusion.

The lead-through drug may be in any form of formulation. For oral administration, the lead-through drug may be in the form of tablets, such as sugar-coated tablets, buccal tablets, coated tablets and chewable tablets, troches, pills, capsules, including powders and soft capsules, granules, suspensions, syrups, including emulsions and dry syrups, and liquids, such as elixirs. For parenteral administration, the lead-through drug may be in the form of injections, inhalants, transdermal tapes, aerosols and suppositories.

The lead-through drug is administered to a subject having a disease caused by PTC, such as DMD, BMD, CF, and cancer. The subject to which the lead-through drug is administered is preferably a vertebrate, and more preferably a mammal. Examples of mammals include humans, chimpanzees and other primates, pigs, and horses, as well as birds such as ducks and chickens. The particularly preferred subject to which the lead-through drug is administered is humans.

The read-through drug according to this embodiment can selectively obtain read-through activity against a stop codon. As shown in the examples below, the type of amino acid inserted at the position corresponding to the PTC in the full-length peptide produced by read-through can be controlled depending on the transfer group.

In another embodiment, a lead-through combination is provided. The lead-through combination comprises the lead-through drug described above, in which R is the above-mentioned substituent, and a compound having the above-mentioned atomic group and donating the atomic group to the substituent. The compound is, for example, the above-mentioned azido-α-CD, β-CD, γ-CD, glucose, G418, amikacin, doxorubicin, and negamycin (azide derivative). The lead-through combination is a combination of the lead-through drug and the compound as active ingredients. The lead-through combination may be a combination drug in which the lead-through drug and the compound are administered in combination.

The term "lead-through combination" includes providing the lead-through drug and compound as a combination drug, as well as providing them as a kit containing each of them as a separate formulation. When the lead-through combination is a combination drug, the lead-through drug and compound can be made into a combination drug by a normal method of mixing multiple components to make a combination drug. The combination drug may further contain any component other than the lead-through drug and compound. The optional component is another pharmacologically acceptable component that can be contained in the lead-through drug described above. The combination drug may be in any form, such as liquid, solid, semi-solid, or powder. The combination ratio of the lead-through drug and compound may be an appropriate ratio that allows each to exert its respective effects.

When the lead-through combination according to this embodiment is a kit of a first formulation containing the lead-through drug and a second formulation containing a compound, the first formulation and the second formulation may be in the same form or in different forms. The first formulation and the second formulation may be formulations having the same administration route or administration method or different administration methods. For example, the first formulation and the second formulation may both be oral administration formulations or parenteral administration formulations, the first formulation may be an oral administration formulation and the second formulation may be a parenteral administration formulation, or the first formulation may be a parenteral administration formulation and the second formulation may be an oral administration formulation. The first formulation may further include any component other than the lead-through drug. The second formulation may further include any component other than the compound. The optional component is another pharmacologically acceptable component that may be included in the lead-through drug described above.

In another embodiment, the use of a nucleic acid probe for the manufacture of a lead-through drug is provided.

The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples.

[Synthesis of Nucleic Acid Probe]
The steps of synthesis of a functional group transfer nucleic acid probe (FT probe) from a probe and a transfer group, synthesis of modified mRNA by utilizing the FT probe, and addition of a compound to the modified mRNA by a click reaction using an azide derivative (X-N ₃ ) are shown in FIG. 1. For the probe, an 11-base-long 2'-OMe RNA was used, which has a base sequence complementary to a base sequence containing a target base in a 131-base-long mRNA to be modified, and in which 4-thio-2'-deoxythymidine (SdT) is incorporated as a nucleic acid pairing with the target adenine of CAA, UAA, UGA, or UAG in the mRNA. When targeting the cytosine of CAA, a 2'-OMe RNA was used in which 6-thio-2'-deoxyguanosine (SdG) is incorporated as a nucleic acid pairing with the target cytosine. The base sequence of the FT probe targeting the terminal adenine of UAA shown in FIG. 1 is shown in SEQ ID NO: 27. The base sequence of the mRNA to be modified, where the codon CAA, UAA, UGA or UAG targeted for modification is NNN, is shown in SEQ ID NO: 28. The base sequence of the mRNA exemplified in Figure 1, where NNN is UAA, is shown in SEQ ID NO: 29. The base sequence of the mRNA to which the transfer group has been transferred is shown in SEQ ID NO: 30.

In the synthesis of the FT probe, the iodine derivative of the transfer group was used. As shown in Figure 1, the transfer group has a pyridinylketovinyl group where R is H or an acetylene group at the end, and has ethylene glycol linkers of different lengths (2EG: n = 2, 3EG: n = 3, 4EG: n = 4).

After synthesis of the FT probe, the FT probe was reacted with mRNA to transfer the transfer group to the 6-amino group of the target adenine. Next, the mRNA was further chemically modified with various molecules by a click reaction between the acetylene group at the end of the transfer group and an azide derivative. Figure 2 shows the structure of the azide derivative. Amikacin, G418, doxorubicin, and negamycin were used as molecules that have been reported to have read-through activity, and glucose, α-CD, β-CD, γ-CD, and pyrene were used as molecules without read-through activity. Azide β-CD (Tokyo Chemical Industry Co., Ltd., A3090), glucose azide (Merck, 712760-100MG), and 1-azidopyrene (Tokyo Chemical Industry Co., Ltd., A2791) were used as azide derivatives of β-CD, glucose, and pyrene, respectively. Azide α-CD and γ-CD, which are azide derivatives of α-CD and γ-CD, were synthesized according to the literature (Tang, W.; Ng, S.C., Facility synthesis of mono-6-amino-6-deoxy-alpha-, beta-, gamma-cyclodextrin hydrochlorides for molecular recognition, chiral separation and drug delivery., Nat Protoc 2008, 3(4), 691-697). The azide derivatives of G418, amikacin, negamycin, and doxorubicin were synthesized by acylation of the amino groups contained in the molecules with succinimidyl 4-azidobutanoate.

In the following, for example, FT probe (UAA ^* , H) means that the nucleic acid paired with the adenine at the end of the mRNA codon UAA is SdT, and the transfer group has a pyridinylketovinyl group. FT probe (UAA ^* , 2EG) means that the nucleic acid paired with the adenine at the end of the mRNA codon UAA is SdT, and the transfer group has 2EG with an acetylene group at its end. FT probe (C ^* AA, H) means that the nucleic acid paired with the cytosine at the mRNA codon CAA is SdG, and the transfer group has a pyridinylketovinyl group.

In this example, the cytosine or terminal adenine of the codon CAA and the second or third adenine in the stop codon (UAA, UAG, UGA) were chemically modified in a sequence- and base-specific manner. The base sequence of the mRNA represented as mRNA (UAA) is the base sequence shown in SEQ ID NO: 29. In the following, for example, mRNA (UAA ^* , 3EG) means an mRNA in which R is 3EG having an acetylene group at the end, and the adenine at the end of the stop codon is modified. mRNA (UAA ^* , 3EG-X) means an mRNA in which a molecule X has been further introduced into the acetylene group at the end of 3EG by a click reaction.

mRNA (NNN) was synthesized by transcription with T7 polymerase using chemically synthesized DNA containing a T7 promoter as a template. In the case of mRNA (CAA) whose base sequence is shown in SEQ ID NO: 31, as shown in FIG. 3, when translation is performed from the start codon to the stop codon, a peptide of 26 amino acid residues (SEQ ID NO: 32) containing a T7 tag and a flag tag is synthesized. In the in vitro translation used in this example, the amino group at the N-terminus of the synthesized peptide is formylated (f-). On the other hand, in the case of mRNA (UAA) where NNN is a PTC, translation is interrupted and a peptide of 17 amino acid residues (17aa; SEQ ID NO: 33) is synthesized. Similarly, 17aa is synthesized when NNN is another PTC, i.e., UGA or UAG.

Even if NNN is a PTC, when read-through occurs due to insertion of a near-cognate tRNA, the peptides synthesized are represented by the one-letter code of the amino acid residues at the positions corresponding to the PTC, 26aa(Y), 26aa(Q), 26aa(K), and 26aa(W). For example, the amino acid sequence of 26aa(Y) is shown in SEQ ID NO:34. The peptides produced by the translation reaction were confirmed by analysis using UPLC/QTOF-MS and by the retention time with synthetic peptide standards.

UPLC conditions Column: ACQUITY UPLC Peptide BEH C18, 300 Å, 1.7 μm, 2.1 × 50 mm
Injection volume: 5 μL
Elution solvent: Solution A water (0.1% HCOOH), Solution B acetonitrile (0.1% HCOOH), B: 5% → 35% / 10 min, 0.2 mL / min, 65 ° C.
MS conditions MS system: Xevo G2-XS QTof
Mass range: 250-5000 Da
Internal standard: leucine-enkephalin Mode: ESI positive, resolution, continuum
Cone voltage: 30V
Capillary voltage: 3 kV
Desolvation temperature: 450°C
Source temperature: 120°C
Desolvation gas flow rate: 800 L/hour Sample solvent: MilliQ

Using a probe (UAA ^* ) having SdT as ^the nucleic acid pairing with the adenine at the end of the mRNA codon UAA, mRNA (UAA ^* , 3EG-βCD) was synthesized as follows. An acetonitrile solution (final concentration 500 μM) of the transfer group iodine body (R=3EG) was added to 100 mM carbonate buffer (pH 10) containing the probe (UAA*) (final concentration 50 mM), and reacted at 25°C for 1 hour to quantitatively prepare an FT probe (UAA ^* , 3EG). The obtained FT probe (UAA ^* , 3EG) and mRNA (UAA) were reacted at 37°C for 1 hour in a HEPES buffer containing NaCl and _NiCl2 . The concentrations of the components in this reaction solution are as follows:
FT probe (UAA ^* , 3EG), 7.5 μM;
mRNA (UAA), 5 μM;
HEPES, 50mM, pH 7.2;
NaCl, 100mM;
NiCl ₂ , 75 μM;

In the click reaction to introduce β-CD, a DMSO solution of azido β-CD, a DMSO solution of TBTA, sodium ascorbate and an aqueous solution of _CuSO4 were added to the mRNA (UAA ^* , 3EG) reaction solution, and the reaction was carried out for 1 hour at 25° C. The final concentrations of this reaction solution were as follows:
13% DMSO β-CD, 400 μM;
TBTA, 160 μM;
Sodium ascorbate, 100 μM;
CuSO ₄ , 200 μM;

FIG. 4 shows HPLC chromatograms tracing the synthesis of the FT probe and the generation of mRNA (UAA ^* , 3EG-βCD). FIG. 4A shows the peak of the probe (UAA ^* ). When a transfer group iodide (R=3EG) was introduced into the probe (UAA ^* ), the FT probe (UAA ^* , 3EG) was obtained as shown in FIG. 4B. When the probe was subsequently mixed with mRNA (UAA), the mRNA (UAA ^* , 3EG) was obtained by a transfer group transfer reaction as shown in FIG. 4C. The reaction solution shown in FIG. 4C contains mRNA (UAA), mRNA (UAA ^* , 3EG), the probe (UAA ^* ) and unreacted FT probe (UAA ^* , 3EG). The mRNA (UAA ^* , 3EG) was isolated from this mixture and used in the translation reaction.

On the other hand, when azide β-CD was added to the reaction solution shown in FIG. 4C to carry out a click reaction, mRNA (UAA ^* , 3EG-βCD) was generated at a retention time of about 11.8 minutes as shown in FIG. 4D. To investigate the effect of chemical modification on the translation reaction, it is necessary to use a highly pure modified mRNA. For this reason, this peak was isolated by HPLC, and a sample containing about 5% unreacted mRNA (UAA) was obtained as shown in FIG. 4E. In order to use the sample in the translation reaction, the sample was concentrated using a centrifugal ultrafiltration filter, and the buffer solution was exchanged before use in the translation reaction. The structure of the modified mRNA was confirmed by measuring the molecular weight using the above-mentioned UPLC/QTOF-MS.

[Translation reaction]
PUREfrex™ 1.0 (Gene Frontier) was used for the translation reaction. The translation reaction was carried out at 37°C for 2 hours at a reaction scale of 20 μL according to the kit protocol. The reaction solution was added to cold dissolution buffer, and Anti-T7-tag pAb-agarose solution was added to this solution and shaken at 4°C for 1 hour. This solution was centrifuged, the supernatant was removed, and the pellet was washed with cold dissolution buffer and then with ultrapure water. The peptide was dissociated by adding 50% acetonitrile-water containing 0.1% formic acid to this pellet, and the supernatant was collected by centrifugation. The collected supernatant was filtered through a spin filter, and the spin filter was washed with 100 μL of MilliQ (4°C, 2500 g, 3 minutes). The filtrate was freeze-dried, and the obtained sample was dissolved in MilliQ and analyzed by UPLC/QTOF-MS.

As a control experiment, pyrene, β-CD, glucose, doxorubicin, amikacin, G418 or negamycin were added to the translation system at the same concentration as the mRNA, and a translation reaction was carried out. As a result, pyrene, β-CD, glucose, doxorubicin and negamycin had no effect on the translation reaction. On the other hand, amikacin and G418 completely inhibited translation. Although it has been reported that amikacin and G418 have read-through activity, it is believed that in this experiment the ribosome inhibition effect as an antibacterial mechanism of action was clearly manifested.

FIG. 5 shows a TIC chromatogram of the translation reaction product of chemically modified mRNA (CAA). As shown in FIG. 5A, it was confirmed that the translation of unmodified mRNA (CAA) produced 26aa (Q) with glutamine incorporated into the CAA codon as the main product. Peak a is a mixture of low molecular weights of 514 and 519, and is a decomposition product derived from agarose of Anti-T7-tag pAb-agarose, which was observed in common in all samples. Peak b is considered to be a peptide 26aa (R) with arginine inserted, which was produced as a by-product by the erroneous insertion of near-cognate tRNA into the CAA code. As shown in FIG. 5B, in mRNA (C ^* AA, 3EG-pyrene), there was almost no change except for the production of non-formylated 26aa (Q) as peak c. There was almost no change in mRNA (CAA ^* , 3EG-pyrene) and mRNA (CAA ^* , 3EG-βCD) except for the production of non-formylated 26aa (Q). It is considered that the non-formylated peptide was produced by incorporation of Met-tRNA due to insufficient production of fMet-tRNA in the system. From the above, it was shown that modification of C and terminal A of CAA has almost no effect on translation.

Next, the effect of modification with UAA as PTC was examined. As shown in FIG. 6A, 17aa was produced by PTC in mRNA (UAA), whereas in the case of mRNA (UAA ^* , H), as shown in FIG. 6B, effective read-through occurred, and 26aa (Y), 26aa (Q), and 26aa (K) were produced by incorporating three types of near-cognate tRNA. The anticodons of tRNAs Y, Q, and K were UAC, CAA, and AAA, respectively, which have two bases in common with UAA. As shown in FIG. 6C, 26aa (Y), 26aa (Q), and 26aa (K) were also produced in mRNA (UAA ^* , 3EG).

When the ethylene glycol linker had α, β, or γ-CD at its end, read-through occurred effectively regardless of the size of the ring, and 26aa(Q) was selectively produced (Figures 6D, 6E, and 6F). As mentioned above, β-CD alone had no effect, so this result indicates that selective chemical modification of PTC allows selective read-through of near-cognate tRNA.

The effect of the length of the ethylene glycol linker on the CD modification of mRNA (CAA) is shown in FIG. 7. Read-through was hardly induced with diethylene glycol (2EG-α,β,γ-CD). The difference between triethylene glycol linker (3EG-α,β,γ-CD) and tetraethylene glycol linker (4EG-α,β,γ-CD) was not large, but the read-through activity of 3EG-CD was slightly higher than that of α-CD and β-CD, and the read-through activity of 4EG-CD was slightly higher than that of γ-CD. In the case of 2EG, the click reaction product, mRNA (CAA ^* , 2EG-α,β,γ-CD), could not be completely separated from mRNA (CAA ^* , 2EG), so a mixture was used for the translation reaction. Read-through activity was also confirmed in a mixture in which mRNA (CAA ^* , 3EG-β-CD) and mRNA (CAA ^* , 3EG) were mixed in the same ratio as the mixture.

As shown in Figures 8A-C, three types of read-through products 26aa(Y), 26aa( ^Q ) and 26aa(K) were obtained with mRNA (UAA ^* , 2EG-glucose), mRNA (UAA ^* , 3EG-glucose) and mRNA (UAA ^* , 4EG-glucose), which had almost the same effect as mRNA (UAA*, 3EG) shown in Figure 6C, and no effect of glucose was observed. On the other hand, G418 and amikacin, which showed inhibition of translation reaction in the control experiment, showed slight read-through activity, although the translation reaction was strongly inhibited, as shown in Figures 8D and 8E.

In control experiments, doxorubicin and negamycin did not affect the translation reaction and 17aa was generated, but when introduced into mRNA, the read-through product 26aa (Y) was selectively generated as shown in Figures 8F and 8G. Since 26aa (Q) was generated in mRNA (UAA ^* , 3EG-α-CD), mRNA (UAA ^* , 3EG-β-CD) and mRNA (UAA ^* , 3EG-γ-CD) (Figures 6D, 6E and 6F), it was shown that the selectivity of the read-through product can be controlled by X introduced into mRNA.

Next, the effect of modification with UAG and UGA as PTC was examined. As shown in Figures 9A and 9B, 17aa was generated from mRNA (UAG) and mRNA (UA ^* G,H), whereas read-through activity was observed from mRNA (UA ^* G,3EG-β-CD), and 26aa (Q) was selectively generated (see Figure 9C). The anticodon CAG of the tRNA for Q shares two bases with UAG. Therefore, it is considered that Gln-tRNA was inserted as a near-cognate tRNA.

As shown in Figure 9D, 17aa was generated from mRNA (UGA), whereas read-through activity was observed from mRNA (UGA ^* , H) and mRNA (UGA ^* , 3EG-β-CD), and 26aa (W) was selectively and efficiently generated (see Figures 9E and 9F). The anticodon UGG of W tRNA shares two bases with UGA. For this reason, it is believed that Trp-tRNA was inserted as a near-cognate tRNA.

UAC (Y), CAA (Q) and AAA (K) were inserted as near-cognate tRNAs without selectivity by chemical modification of the A at the end of UAA with a pyridine keto group (see Figure 6B). In contrast, UGG (W) was selectively inserted by chemical modification of the A at UGA with a pyridine keto group (see Figure 9E). From these findings, it is possible that the pyridine keto group inhibits the formation of a complex between RF2 and PTC. To confirm this, the RF concentration dependency of read-through activity was examined. In this study, translation of mRNA containing UGA, an RF2-dependent PTC, and UAG, an RF1-dependent termination codon, was performed using Costomized PUREfrex (trademark) 1.0 (Gene Frontier Co., Ltd.) that does not contain RF2, and the concentration of added RF2 was changed to examine the relationship between the production ratio of 17aa and 26aa and the RF2 concentration.

As shown in Figure 10, when the RF2 concentration was low, unmodified mRNA (UGA) produced 26aa (W) in which Trp-tRNA was selectively incorporated, but as the RF2 concentration increased, the amount of 26aa (W) produced decreased and the amount of 17aa produced increased. In contrast, when the RF2 concentration was increased in modified mRNA (UGA ^* , H), there was almost no effect on the amount of 26aa (W) produced. This confirmed that modification of the 6th position of adenine in UGA strongly inhibited the formation of a complex between RF2 and PTC in the ribosome.

The insertion of three types of near-cognate tRNAs into mRNA (UAA ^* , H) can also be interpreted as being due to the inhibition of complex formation between PTC and RF1. Furthermore, since CAA (Q) was selectively inserted into mRNA (UAA ^* , 3EG-β-CD), it is expected that β-CD exerts some selective effect on the complex formation between Gln-tRNA and UAA.

The above-described embodiment is intended to explain the present invention, and does not limit the scope of the present invention. In other words, the scope of the present invention is indicated by the claims, not by the embodiment. Furthermore, various modifications made within the scope of the claims and within the scope of the meaning of the invention equivalent thereto are considered to be within the scope of the present invention.

The present invention is useful as a medicine for diseases caused by premature stop codons.

Claims (8)

Contains a nucleic acid probe,
The nucleic acid probe comprises:
a base sequence complementary to an mRNA to be modified, and a reactive base derivative at a position complementary to an adenine contained in a termination codon to be targeted for modification in the mRNA;
transferring a transfer group contained in the reactive base derivative to the 6-amino group of the adenine;
Lead-through drugs. The reactive base derivative is
As shown in formula I:
The lead-through drug of claim 1 .
(In formula I, R is H or a substituent that includes a linker terminated with an alkynyl group.) R is a substituent containing a linker having an alkynyl group at its terminal end,
an atomic group is added to the substituent via the alkynyl group in the transfer group transferred to the mRNA;
The lead-through drug of claim 2. As the atomic group, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, glucose, G418, amikacin, doxorubicin or negamycin is added to the substituent via a hydroxy group or an amino group;
The lead-through drug of claim 3 . The adenine contained in the stop codon is
A at the end of UAA, A at the end of UAG, or A at the end of UGA,
The linker is
A triethylene glycol linker or a tetraethylene glycol linker,
As the atomic group, α-cyclodextrin, β-cyclodextrin or γ-cyclodextrin is added to the substituent via a hydroxy group;
The lead-through drug of claim 4. The adenine contained in the stop codon is
The terminal A of UAA,
As the atomic group, doxorubicin or negamycin is added to the substituent via an amino group;
The lead-through drug of claim 4. A lead-through drug according to any one of claims 3 to 6;
a compound having the atomic group and donating the atomic group to the substituent;
A lead-through combination comprising: 1. Use of a nucleic acid probe for the manufacture of a read-through drug, comprising:
The nucleic acid probe comprises:
a base sequence complementary to an mRNA to be modified, and a reactive base derivative at a position complementary to an adenine contained in a termination codon to be targeted for modification in the mRNA;
transferring a transfer group contained in the reactive base derivative to the 6-amino group of the adenine;
Use of nucleic acid probes.