WO2023120658A1 - 酵素、複合体、組換えベクター、遺伝性疾患治療薬及びポリヌクレオチド - Google Patents

酵素、複合体、組換えベクター、遺伝性疾患治療薬及びポリヌクレオチド Download PDF

Info

Publication number
WO2023120658A1
WO2023120658A1 PCT/JP2022/047410 JP2022047410W WO2023120658A1 WO 2023120658 A1 WO2023120658 A1 WO 2023120658A1 JP 2022047410 W JP2022047410 W JP 2022047410W WO 2023120658 A1 WO2023120658 A1 WO 2023120658A1
Authority
WO
WIPO (PCT)
Prior art keywords
rna
enzyme
uridine
protein
coat protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2022/047410
Other languages
English (en)
French (fr)
Japanese (ja)
Inventor
俊文 塚原
ルチカ
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Japan Advanced Institute of Science and Technology
Original Assignee
Japan Advanced Institute of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Japan Advanced Institute of Science and Technology filed Critical Japan Advanced Institute of Science and Technology
Priority to EP22911360.0A priority Critical patent/EP4455288A4/en
Priority to JP2023569543A priority patent/JPWO2023120658A1/ja
Priority to CN202280092054.8A priority patent/CN118715322A/zh
Priority to US18/723,452 priority patent/US20250302993A1/en
Publication of WO2023120658A1 publication Critical patent/WO2023120658A1/ja
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1096—Transferases (2.) transferring nitrogenous groups (2.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y206/00—Transferases transferring nitrogenous groups (2.6)
    • C12Y206/01—Transaminases (2.6.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04005—Cytidine deaminase (3.5.4.5)

Definitions

  • the present invention relates to enzymes, complexes, recombinant vectors, genetic disease therapeutic agents and polynucleotides for converting uridine in RNA to cytidine.
  • a nonsense mutation is a mutation that changes a codon encoding an amino acid to a stop codon.
  • a stop codon caused by a mutation is called an immature stop codon, and nothing after the immature stop codon is translated.
  • Diseases caused by nonsense mutations are referred to as nonsense-mutated genetic diseases and vary, for example, cystic fibrosis (CF) and Duchenne muscular dystrophy (DMD).
  • CF cystic fibrosis
  • DMD Duchenne muscular dystrophy
  • Patent Document 1 discloses an aminoglycoside compound.
  • Aminoglycoside compounds are concerned about side effects such as hearing loss and aggravating the symptoms of patients with renal impairment.
  • side effects such as hearing loss and aggravating the symptoms of patients with renal impairment.
  • not only the target immature stop codon but also the original stop codon are often read through to add an extra C-terminal peptide to the normal protein, and side effects due to the added peptide are also a concern.
  • Genome editing is expected as a treatment for hereditary diseases. Genome editing is a technique for inducing mutation by repairing DNA by site-specific double-strand cleavage of genomic DNA and then non-homologous end joining or homologous recombination. However, at present, it is very difficult to perform accurate genome editing in all target cells in vivo, so it is necessary to select cells whose genomes have been correctly edited after genome editing is performed. Moreover, there are many ethical concerns with genome editing. Although genome editing is a method suitable for ex vivo or fertilized eggs, it is difficult to apply to the whole body of a patient.
  • adenosine deaminase 1 (ADAR1) that acts on RNA and the RNA-binding coat protein from the MS2 phage (MS2 coat protein) and RNA that specifically binds to it to correct guanosine to adenosine mutations in genes
  • a method using the MS2 system containing (MS2 RNA) is disclosed in Non-Patent Document 1.
  • a guide RNA is composed of a complementary strand of the target RNA, and an artificial enzyme complex in which MS2 RNA, MS2 coat protein and ADAR1 are sequentially bound to the guide RNA. to use.
  • ADAR1 is induced in the vicinity of the target RNA by the guide RNA.
  • a guide RNA in which the base corresponding to the adenosine to be corrected is cytidine
  • deamination by ADAR1 is facilitated because adenosine is mismatched with cytidine in the guide RNA.
  • Patent Document 2 discloses a method for biosynthesizing a full-length mRNA protein by modifying the nucleobase that constitutes the premature stop codon and allowing read-through. The method is based on read-through of the premature stop codon when a functional group is introduced into the nucleobases that constitute the premature stop codon by methylation or halogenation.
  • stop codons There are only three types of stop codons: UAA, UAG and UGA. If the uridine can be converted to another base, the nonsense mutation can be a codon encoding an amino acid.
  • RNA editing of mRNA from cytidine to uridine is known.
  • the PPR (pentatriceptide repeat) protein of Physcomitrium patens which binds to RNA in a nucleotide sequence-specific manner, has a PPR motif repeat with two ⁇ -helical structures consisting of about 35 amino acids.
  • PPR-DYW which has an E (extension) domain and a DYW (Asp-Tyr-Trp) domain at the C-terminus, has deaminase activity, catalyzes the deamination of cytidine, and converts cytidine. known to convert to uridine.
  • the present invention has been made in view of the above circumstances, and provides enzymes, complexes, recombinant vectors, hereditary disease therapeutic agents, and polynucleotides capable of converting uridine generated in mRNA by mutation into cytidine. for the purpose.
  • RNA editing from uridine to cytidine is observed in hornwort.
  • the present inventors have conducted extensive research, identified an enzyme that catalyzes the reaction, and completed the present invention.
  • the enzyme according to the first aspect of the present invention is It has activity to convert uridine in RNA to cytidine.
  • the enzyme according to the first aspect of the present invention is (i) a region having the amino acid sequence shown in SEQ ID NO: 1 or 2, or (ii) a region having an amino acid sequence with 90% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 2; including, You can do it.
  • the composite according to the second aspect of the present invention is the enzyme according to the first aspect of the present invention; a sequence recognition module that causes the enzyme to specifically act on uridine generated in mRNA by mutation; including.
  • the uridine is a uridine contained in a premature stop codon formed by a nonsense mutation; You can do it.
  • the sequence recognition module is a guide RNA complementary to at least a portion of the target RNA containing uridine; an RNA-binding coat protein from the MS2 phage; MS2 RNA that specifically binds to the RNA-binding coat protein; including
  • the guide RNA is A fusion RNA with the MS2 RNA
  • the enzyme is a fusion protein with the RNA-binding coat protein, wherein the fusion protein binds to the guide RNA via binding of the RNA-binding coat protein to the MS2 RNA; You can do it.
  • the recombinant vector according to the third aspect of the present invention is A fusion protein of the enzyme according to the first aspect of the present invention and an RNA-binding coat protein derived from MS2 phage, MS2 RNA that specifically binds to the RNA-binding coat protein, and uridine generated in mRNA by mutation.
  • a fusion RNA with a guide RNA complementary to at least a portion of the target RNA comprising A complex in which the enzyme binds to the guide RNA through binding between the RNA-binding coat protein and the MS2 RNA is expressed and formed in the cell.
  • the hereditary disease therapeutic drug according to the fourth aspect of the present invention is The complex according to the second aspect of the present invention, or the recombinant vector according to the third aspect of the present invention.
  • the polynucleotide according to the fifth aspect of the present invention is It encodes the enzyme according to the first aspect of the present invention.
  • the polynucleotide according to the sixth aspect of the present invention is A fusion protein of the enzyme according to the first aspect of the present invention and an RNA-binding coat protein derived from MS2 phage, MS2 RNA that specifically binds to the RNA-binding coat protein, and uridine generated in mRNA by mutation.
  • a fusion RNA with a guide RNA complementary to at least a portion of a target RNA comprising Code a complex containing
  • uridine generated in mRNA by mutation can be converted into cytidine.
  • FIG. 2 is a diagram showing the configuration of a construct according to Example 1;
  • FIG. 1 is a diagram showing base sequences detected in Example 1.
  • FIG. FIG. 10 is a diagram for explaining a construct according to Example 2;
  • A is a diagram showing the region contained in each defective protein.
  • B is a schematic diagram of an artificial RNA-editing enzyme complex.
  • FIG. 2 shows base sequences detected in Example 2.
  • the enzyme according to this embodiment has the activity of converting uridine in RNA into cytidine. More specifically, the enzyme catalyzes a reaction that transfers an amino group to uracil, which is the nucleobase of uridine, to produce cytidine.
  • the enzyme is not particularly limited as long as it has the activity of converting uridine in RNA into cytidine.
  • an enzyme according to the present embodiment is an aminotransferase derived from hornwort.
  • the E2 (also referred to as E+, hereinafter referred to as "E2") domain of the PPR protein is essential for the deaminase activity of PPR-DYW.
  • the E2 domain is essential for the transamination activity of the enzyme according to this embodiment.
  • the amino acid sequence of the E2 domain is exemplified in SEQ ID NO:3, for example.
  • the GRP domain linked to the E2 domain is an enzyme that catalyzes the reaction of amino group transfer to uridine to produce cytidine, as shown in Example 1 below.
  • the amino acid sequence of the E2-GRP domain is shown, for example, in SEQ ID NO:1 or SEQ ID NO:2.
  • the enzyme may be an E2-GRP-like protein of a plant other than hornwort, or an enzyme obtained by modifying cytidine deaminase to have transamination activity, as long as it has the activity of converting uridine into cytidine.
  • it may be an enzyme modified to act on uridine in RNA from an enzyme that transfers amino groups to free uridine.
  • the enzyme according to the present embodiment has an activity to convert uridine in RNA to cytidine (hereinafter also referred to as "uridine-cytidine conversion activity”) and includes the following regions (i) or (ii): may be (i) a region having the amino acid sequence shown in SEQ ID NO: 1 or 2; (ii) a region having an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1 or 2;
  • sequence identity in (ii) above is, for example, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.
  • the enzyme according to the present embodiment has a uridine-cytidine conversion activity and is an amino acid sequence obtained by substituting, inserting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO: 1 or 2. It may be a region having The number of substituted, inserted, deleted or added amino acids is one or several. Several is, for example, 20 or less, 15 or less, or 10 or less. Preferably, several is any within the range of 2-9.
  • the uridine-cytidine conversion activity of the enzyme according to the present embodiment is obtained by, for example, expressing a target RNA containing uridine in Escherichia coli or the like and the enzyme, allowing the enzyme to act on the target RNA, and determining the base sequence of the target RNA. can be evaluated by If uridine in the nucleotide sequence is converted to cytidine, the enzyme has uridine-cytidine conversion activity.
  • a known sequence recognition module that allows the enzyme to act specifically on uridine present in a predetermined region of the target RNA may be used. The sequence recognition module will be described later.
  • the enzyme according to the present embodiment has uridine-cytidine conversion activity, it can be used to convert uridine generated in RNA by mutation into cytidine.
  • a polynucleotide encoding the above enzyme is provided.
  • the complex according to the present embodiment includes the enzyme according to the first embodiment, and a sequence recognition module that causes the enzyme to act specifically on uridine generated in mRNA due to mutation.
  • the uridine is the uridine contained in the premature stop codon formed by nonsense mutation.
  • sequence recognition module is not particularly limited as long as it allows the enzyme to act on uridine present in a predetermined region of the mRNA as the target RNA.
  • a sequence recognition module may be a single molecule or a complex of multiple molecules.
  • a sequence recognition module includes at least one of a protein and nucleic acid that binds sequence-specifically to mRNA. More specifically, examples of the sequence recognition module include CRISPR-dCAS system using dCas13 with inactivated nucleic acid cleaving ability, zinc finger motif, TAL effector, PPR protein, DNA and peptide nucleic acid (PNA). be.
  • the CRISPR-dCas system uses a dCas protein that has had its nuclease and nickase activities eliminated, and a guide RNA.
  • the guide RNA consists of a base sequence that forms a base pair with the complementary strand of the target sequence, which corresponds to CRISPR RNA (crRNA), and a base sequence that functions as a transactivating crRNA (tracrRNA) and serves as a scaffold for dCas protein binding.
  • CRISPR RNA CRISPR RNA
  • tracrRNA transactivating crRNA
  • a dCas-guide RNA complex is formed by base-pairing of the guide RNA to the complementary strand of the target sequence.
  • a zinc finger motif is a combination of multiple different Cys2His2-type zinc finger units.
  • Zinc finger motifs can be produced by known methods such as the modular assembly method, the OPEN method, the CoDA method and the E. coli one-hybrid method.
  • the above enzyme can be made to act specifically on uridine.
  • a TAL effector has a repeating structure of modules of about 34 amino acids, and the 12th and 13th amino acid residues of one module determine binding stability and base specificity.
  • TAL effectors REAL method, Golden Gate method and the like have been established.
  • the above enzyme can be caused to specifically act on uridine.
  • a PPR protein can be configured to recognize a specific base sequence by a sequence of PPR motifs that recognize one nucleobase.
  • the above enzyme can be made to act specifically on uridine.
  • N-(2-aminoethyl)glycine not sugar, is linked by an amide bond.
  • the purine and pyrimidine rings corresponding to nucleobases in PNA are attached to the main chain via a methylene group and a carbonyl group.
  • the enzyme and the sequence recognition module may be linked by a compound, a linker, or the like via a covalent bond.
  • a linker is a chemical group or molecule that connects the enzyme and the sequence recognition module.
  • a linker may connect the enzyme with proteins and nucleic acids that bind to mRNA in a sequence-specific manner.
  • a linker is, for example, positioned between two groups, molecules, or other moieties and linked to each by a covalent bond.
  • a linker may be a single amino acid or multiple amino acids.
  • a linker may be an organic molecule, group, polymer, or the like.
  • the linker consists of, for example, 3-200 amino acids, such as 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20, 30 ⁇ 35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150 or 150 There may be ⁇ 200 peptides.
  • the genome of the MS2 phage is a single-stranded RNA that functions as a +-strand mRNA.
  • MS2 phage infects Escherichia coli, negative-strand RNA is synthesized following translation of the genes required for proliferation of MS2 phage. Using this ⁇ strand RNA as a template, + strand RNA is synthesized.
  • MS2 phage synthesizes various proteins of MS2 based on RNA, and among them, the MS2 coat protein has the property of binding upstream of replication genes.
  • the MS2 phage has the characteristic that the viral coat protein binds to the viral mRNA that serves as a template for translation, that is, the genomic RNA.
  • protein and RNA can be specifically linked.
  • the sequence recognition module includes guide RNA, MS2 coat protein, and MS2 RNA.
  • a guide RNA is an RNA that is complementary to at least part of an mRNA (target sequence) that contains uridine that has been mutated in the mRNA. If the nucleobase U to be converted is a "target nucleobase", the base sequence of the guide RNA may be composed of a base sequence complementary to all bases of at least a part of the base sequence including the target nucleobase of the mRNA. However, as long as the guide RNA hybridizes to the target sequence, the guide RNA may contain mismatches to the target sequence, that is, it may be a base sequence complementary to bases excluding one or several bases of the target sequence. .
  • the guide RNA contains mismatches, it is preferred that only the nucleobases in the guide RNA that correspond to the target nucleobase are mismatched.
  • the nucleobase corresponding to the target nucleobase in the guide RNA is a base other than adenine (A).
  • the guide RNA may be a fusion RNA with MS2 RNA (guide RNA-MS2 RNA).
  • a fusion RNA can be prepared based on the base sequence of the guide RNA.
  • the 3' end or 5' end of the guide RNA and one end of the MS2 RNA may be directly linked, or may be linked indirectly via a linker sequence or the like.
  • the above enzyme may be a fusion protein (MS2 coat protein-enzyme) with MS2 coat protein.
  • the enzyme is bound to the guide RNA through binding between the MS2 coat protein and the MS2 RNA.
  • the C-terminus of the MS2 coat protein and the N-terminus of the enzyme may be directly linked, or indirectly via a linker peptide or the like. Fusion proteins can be generated based on the genetic sequences of the MS2 coat protein and the enzyme.
  • MS2 RNA and MS2 coat protein have strong binding properties
  • the complex according to the present embodiment can cause an enzyme that converts uridine to cytidine to specifically act on the target uridine by means of the sequence recognition module.
  • mutated mRNA can be repaired by targeting the uridine produced by the C>T point mutation.
  • Nonsense mutated mRNA can be converted to allow synthesis of full-length protein.
  • the complex according to the present embodiment targets the uridine of the immature stop codon formed by the nonsense mutation, so that the nonsense-mutated RNA can be converted into a full-length protein. . This allows the production of full-length proteins.
  • a polynucleotide encoding a complex containing the fusion protein and the fusion RNA is provided.
  • the sequence recognition module contains a nucleic acid that binds sequence-specifically to mRNA
  • the nucleic acid may be DNA.
  • the target sequence recognized by the nucleic acid may not contain the target nucleic acid base as long as it is in the vicinity of the target nucleic acid base.
  • the vicinity is, for example, counted from the target nucleic acid base in the direction of the 5' end of the mRNA, from the 2nd to 10th, 2nd to 8th, 2nd to 6th or 2nd to 4th bases in the direction of the 5' end.
  • a base sequence up to a predetermined number of bases may be used as the target sequence, or the 2nd to 10th, 2nd to 8th, 2nd to 6th or 2nd to 4th counted from the target nucleic acid base in the direction of the 3' end of the mRNA.
  • a target sequence may be a nucleotide sequence extending from the base to a predetermined number of bases in the direction of the 3' end.
  • the sequence recognition module contains a protein that binds sequence-specifically to mRNA
  • the nucleotide sequence in the mRNA recognized by the protein is 2 to 10 counted from the target nucleic acid base in the 5' end direction of the mRNA, 2 to It may be a base sequence from the 8th, 2nd to 6th or 2nd to 4th bases to a predetermined number of bases in the direction of the 5' end, or 2 to 2 to the target nucleic acid base in the direction of the 3' end of the mRNA. It may be a base sequence from the 10th, 2nd to 8th, 2nd to 6th or 2nd to 4th bases to a predetermined number of bases in the direction of the 3' end.
  • Mutants of the MS2 coat protein may also be used as long as they bind sequence-specifically to the target mRNA.
  • known RNA-binding proteins such as the ⁇ N system and RNA other than the MS2 system may be used as the sequence recognition module.
  • the ⁇ N system utilizes the ⁇ N22 peptide and the base sequence (Box-B) of RNA to which the ⁇ N22 peptide specifically binds.
  • Variants of the ⁇ N22 peptide may be used as long as they exhibit specific binding to the target mRNA.
  • RNA-binding proteins include PP7 bacteriophage coat protein, Mu bacteriophage Com protein, stem-loop binding protein (SLBP), fragile X mental retardation syndrome-related protein 1 (FXR1), proteins derived from bacteriophages, such as AP205, BZ13, f1, f2, fd, fr, ID2, JP34/GA, JP501, JP34, JP500, KU1, M11, M12, MX1, NL95, PP7, ⁇ Cb5, ⁇ Cb8r, ⁇ Cb12r, ⁇ Cb23r, Q ⁇ , R17, SP- ⁇ , TW18, TW19 and VK, fragments thereof, or derivatives thereof.
  • SLBP stem-loop binding protein
  • FXR1 fragile X mental retardation syndrome-related protein 1
  • the recombinant vector according to the present embodiment expresses and forms the complex using the MS2 system according to the second embodiment in cells.
  • the recombinant vector contains the fusion protein and the fusion RNA, and expresses and forms a complex in which the enzyme binds to the guide RNA through binding between the MS2 coat protein and the MS2 RNA in the cell. .
  • the recombinant vector comprises, based on the gene sequence of the enzyme, a first polynucleotide encoding a fusion protein of the enzyme and the MS2 protein, and a second polynucleotide encoding the guide RNA and MS2 RNA. It may be mounted in one recombinant vector, or the first polynucleotide and the second polynucleotide may be mounted in separate recombinant vectors.
  • the nucleotide sequences of the first polynucleotide and the second polynucleotide can be determined based on the encoding amino acid sequence or RNA nucleotide sequence.
  • a polynucleotide can be prepared based on the determined base sequence.
  • the nucleotide sequences of the first polynucleotide and the second polynucleotide can be introduced into various vectors using restriction enzymes and DNA ligase, or plasmid construction kits based on homologous recombination.
  • the fusion protein may contain a linker sequence interposed between the enzyme and the MS2 protein. Also, there may be a base sequence intervening between the guide RNA and the MS2 RNA.
  • the recombinant vector it is possible to express the MS2 RNA linked to the fusion protein of the above enzyme and the MS2 protein and the guide RNA. This allows an enzyme that converts uridine to cytidine to act specifically on the target nucleobase in intracellular mRNA.
  • the recombinant vector can be used to repair mutated mRNA or to convert nonsense-mutated mRNA into a full-length protein.
  • the recombinant vector allows the complex to be expressed and formed in cells.
  • DNA which is more stable than RNA, it is possible to improve the stability in storage and the like compared to the above-described complex containing the guide RNA, and the handling at the time of use is simplified.
  • the hereditary disease therapeutic drug according to the present embodiment includes the complex according to the second embodiment or the recombinant vector according to the third embodiment.
  • Inherited diseases are diseases caused by nonsense mutations.
  • a genetic disease may be a disease caused by a T>C point mutation.
  • the genetic disease is a nonsense-mutant genetic disease, including CF and DMD.
  • the hereditary disease therapeutic drug according to the present embodiment can be manufactured by a known method.
  • the hereditary disease therapeutic drug may contain other pharmacologically acceptable components in addition to the above complex or recombinant vector as an active ingredient.
  • Hereditary disease therapeutic agents include, for example, complexes or recombinant vectors and pharmacologically acceptable carriers.
  • Pharmaceutically acceptable carriers are various organic or inorganic carrier substances used as pharmaceutical ingredients.
  • Pharmaceutically acceptable carriers are, for example, excipients, lubricants, binders and disintegrants in solid formulations, or solvents, solubilizers, suspending agents, tonicity agents, and buffers in liquid formulations. and an analgesic. Additives such as preservatives, antioxidants, coloring agents and sweetening agents may be added to the hereditary disease therapeutic agent, if necessary.
  • the hereditary disease therapeutic agent according to the present embodiment is administered to humans and animals.
  • Animals are preferably mammals, more specifically dogs, cats, cows, pigs, horses, sheep and deer.
  • the dose of the hereditary disease therapeutic drug according to the present embodiment is appropriately determined according to the subject's sex, age, weight, symptoms, and the like.
  • the hereditary disease therapeutic agent is administered so that the complex or recombinant vector is in an effective amount.
  • An effective amount is that amount of the conjugate or recombinant vector necessary to obtain the desired result, the amount necessary to slow, inhibit, prevent, reverse or cure the condition being treated or treated. be.
  • the hereditary disease therapeutic agent is preferably used as an external preparation, an injection, or an orally administered preparation.
  • the hereditary disease therapeutic agent causes an enzyme that converts uridine to cytidine to act specifically on the target nucleobase in mRNA, thereby converting nonsense-mutated mRNA into a full-length protein that can be synthesized. and the full-length protein can be produced. Therefore, the drug for treating hereditary diseases is effective in treating diseases caused by nonsense mutations.
  • a method for treating, ameliorating, or preventing a genetic disease in a subject by administering the complex according to Embodiment 2 above or the recombinant vector according to Embodiment 3 above to the subject is provided.
  • Another embodiment is the use of the above complexes or recombinant vectors for treating, ameliorating or preventing genetic diseases.
  • the above conjugates or recombinant vectors are provided for use as genetic disease therapeutics.
  • Another embodiment is the use of the above complex or recombinant vector for the production of a drug for treating hereditary diseases.
  • the complex according to Embodiment 2 above or the recombinant vector according to Embodiment 3 above may be used as a reagent for experiments such as in vitro, in vivo, and ex vivo.
  • Example 1 Detection of RNA Editing Activity of DYW Homologs Using PPR56 As shown in FIG. 1, a construct with a GRP domain was prepared.
  • the Physcomitrium patens PPR56 gene (NCBI identification number: LOC112295756) was inserted downstream of the MPB promoter, and the GRP domain was inserted as a DYW homolog in place of the DYW domain of PPR56.
  • a targeted Nad4 gene was inserted further downstream.
  • PPR56 binds the Nad4 gene transcribed mRNA and brings the GRP domain into close proximity to the target nucleobase.
  • a mutation was introduced at the corresponding position in the Nad4 gene so that the target nucleic acid base was U.
  • SEQ ID NO: 4 shows the nucleotide sequence of the mutated Nad4 gene.
  • BL21, Rosetta 2 (DE3) was transformed by a conventional method to obtain a strain.
  • Two clones (GRP-g16507 (SEQ ID NO: 1) and GRP-g63 (SEQ ID NO: 2)) into which the GRP domain was inserted were obtained.
  • Colonies were preincubated with Luria Broth, 50 ⁇ M kanamycin and 30 ⁇ M chloramphenicol. 40 ⁇ L of preculture was inoculated into 4 mL of the same medium and cultured at 37° C. to an OD 600 of 0.4-0.6.
  • FIG. 2 shows part of the base sequence of DNA obtained by reverse transcription of RNA to DNA.
  • GRP-g63 and GRP-g16507 detected only a thymine (T) peak at the position corresponding to the target nucleobase, but in the presence of asparagine, GRP-g63 and GRP-g16507 Cytosine (C) peaks of 61.45% and 67.50%, respectively, were detected overlapping the thymine (T) peak.
  • the GRP domain was shown to convert the target nucleobase U to C in some mRNAs of the Nad4 gene.
  • Example 2 Detection of RNA Editing Activity of DYW Homolog Using PPR56 Cells used in the experiment constitutively have a gene (BFP) in which the fluorescent color is changed from green to blue instead of green due to mutation of the EGFP gene from C to T.
  • BFP gene in which the fluorescent color is changed from green to blue instead of green due to mutation of the EGFP gene from C to T.
  • HEK293 cells differentially expressing
  • a guide RNA having a nucleotide sequence complementary to the nucleotide sequence near the mutated base was prepared. The nucleotide sequence of the guide RNA is shown in SEQ ID NO:7.
  • defective proteins P2-DYW, L2-DYW, S2-DYW, E1- DYW, E2-DYW and DYW
  • a deaminase plasmid was prepared that expresses a gene encoding a fusion protein between the defective protein substituted with PPR56/DYW shown in FIG. 3B and the MS2 coat protein.
  • the amino acid sequences of P2-DYW, L2-DYW, S2-DYW, E1-DYW, E2-DYW and DYW are shown in SEQ ID NOs: 8, 9, 10, 11, 12 and 13 respectively.
  • HEK293 cells stably expressing the above BFP were seeded at a density of 5.5 ⁇ 10 5 cells/well, and the culture medium was removed so that the volume per well was 0.5 mL before transfection. 4 mL of fresh FBS-containing DMEM was added. Next, 50 ⁇ L of Opti-MEM and 500 ng of plasmid (250 ng deaminase plasmid and 250 ng guide RNA) were mixed, incubated at room temperature for 20 minutes, and added to each well. 48 hours after transfection, green fluorescence was observed under a fluorescence microscope, and total RNA was extracted from the transfected cells. After reverse transcription of the extracted total RNA, base sequence analysis was performed to confirm the base sequence of the BFP gene or EGFP gene in the cells into which each defective protein gene had been introduced.
  • FIG. 4 shows the results of RNA editing by introduction of defective protein genes.
  • the deamination activity of DYW required the E2 domain of the PPR protein. The same was confirmed for the GRP domain.
  • the present invention is useful for pharmaceuticals or research reagents.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
PCT/JP2022/047410 2021-12-24 2022-12-22 酵素、複合体、組換えベクター、遺伝性疾患治療薬及びポリヌクレオチド Ceased WO2023120658A1 (ja)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP22911360.0A EP4455288A4 (en) 2021-12-24 2022-12-22 ENZYME, COMPOSITE, RECOMINATING VECTOR, MEDICINE FOR HEREDITARY DISEASE AND POLYNUCLEOTIDE
JP2023569543A JPWO2023120658A1 (https=) 2021-12-24 2022-12-22
CN202280092054.8A CN118715322A (zh) 2021-12-24 2022-12-22 酶、复合体、重组载体、遗传性疾病治疗药以及多核苷酸
US18/723,452 US20250302993A1 (en) 2021-12-24 2022-12-22 Enzyme, complex, recombinant vector, therapeutic agent for genetic disorder, and polynucleotide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2021210380 2021-12-24
JP2021-210380 2021-12-24

Publications (1)

Publication Number Publication Date
WO2023120658A1 true WO2023120658A1 (ja) 2023-06-29

Family

ID=86902714

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/047410 Ceased WO2023120658A1 (ja) 2021-12-24 2022-12-22 酵素、複合体、組換えベクター、遺伝性疾患治療薬及びポリヌクレオチド

Country Status (5)

Country Link
US (1) US20250302993A1 (https=)
EP (1) EP4455288A4 (https=)
JP (1) JPWO2023120658A1 (https=)
CN (1) CN118715322A (https=)
WO (1) WO2023120658A1 (https=)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025094924A1 (ja) * 2023-10-30 2025-05-08 株式会社GeCoRT 酵素、酵素複合体、および、これらの利用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009532461A (ja) 2006-04-03 2009-09-10 テクニオン リサーチ アンド ディベロップメント ファウンデーション リミテッド 新規なアミノグリコシド類および遺伝子疾患の治療におけるその使用
JP2020124155A (ja) 2019-02-05 2020-08-20 国立大学法人北陸先端科学技術大学院大学 タンパク質を生合成させる方法および未成熟終止コドンの修飾方法
WO2021201198A1 (ja) * 2020-03-31 2021-10-07 エディットフォース株式会社 標的ｒｎａを編集する方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2784157T3 (da) * 2011-10-21 2019-10-21 Univ Kyushu Nat Univ Corp Designfremgangsmåde til et rna-bindende protein under anvendelse af ppr-motiv og anvendelse deraf

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009532461A (ja) 2006-04-03 2009-09-10 テクニオン リサーチ アンド ディベロップメント ファウンデーション リミテッド 新規なアミノグリコシド類および遺伝子疾患の治療におけるその使用
JP2020124155A (ja) 2019-02-05 2020-08-20 国立大学法人北陸先端科学技術大学院大学 タンパク質を生合成させる方法および未成熟終止コドンの修飾方法
WO2021201198A1 (ja) * 2020-03-31 2021-10-07 エディットフォース株式会社 標的ｒｎａを編集する方法

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
"NCBI", Database accession no. LOC112295756
BHAKTA SONALI; TSUKAHARA TOSHIFUMI: "Double MS2 guided restoration of genetic code in amber (TAG), opal (TGA) and ochre (TAA) stop codon", ENZYME AND MICROBIAL TECHNOLOGY, STONEHAM, MA, US, vol. 149, 11 June 2021 (2021-06-11), US , XP086702657, ISSN: 0141-0229, DOI: 10.1016/j.enzmictec.2021.109851 *
GERKE PHILIPP, SZÖVÉNYI PÉTER, NEUBAUER ANNA, LENZ HENNING, GUTMANN BERNARD, MCDOWELL ROSE, SMALL IAN, SCHALLENBERG‐RÜDINGER MAREI: "Towards a plant model for enigmatic U‐to‐C RNA editing: the organelle genomes, transcriptomes, editomes and candidate RNA editing factors in the hornwort Anthoceros agrestis", NEW PHYTOLOGIST, WILEY-BLACKWELL PUBLISHING LTD., GB, vol. 225, no. 5, 1 March 2020 (2020-03-01), GB , pages 1974 - 1992, XP093073777, ISSN: 0028-646X, DOI: 10.1111/nph.16297 *
MD TA AZAD: "Site-directed RNA editing by adenosine deaminase acting on RNA for correction of the genetic code in gene therapy", GENE THERAPY, vol. 24, 2017, pages 779 - 786, XP037648995, DOI: 10.1038/gt.2017.90
RUCHIKA KYUSHU, TSUKAHARA TOSHIFUMI, TAKENAKA MIZUKI, NAKAMURA TAKAHIRO: "P-023: Artificial deaminase system with plant derived "DYW" type PPR protein", PROCEEDINGS OF THE ANNUAL MEETING OF THE RNA SOCIETY OF JAPAN; 2022-07-20-22, RNA SOCIETY OF JAPAN, JP, 20 July 2022 (2022-07-20) - 22 July 2022 (2022-07-22), JP, pages 128, XP009546507 *
See also references of EP4455288A4

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025094924A1 (ja) * 2023-10-30 2025-05-08 株式会社GeCoRT 酵素、酵素複合体、および、これらの利用

Also Published As

Publication number Publication date
US20250302993A1 (en) 2025-10-02
EP4455288A4 (en) 2025-12-31
EP4455288A1 (en) 2024-10-30
CN118715322A (zh) 2024-09-27
JPWO2023120658A1 (https=) 2023-06-29

Similar Documents

Publication Publication Date Title
US20210087568A1 (en) Modified Guide RNAs for Gene Editing
JP7814052B2 (ja) 標的ゲノム修飾のための高度に効率的なｒｎａ－アプタマー動員媒介性ｄｎａ塩基エディターおよびそれらの使用
TW202113074A (zh) 工程化之ｃａｓｘ系統
CN113631708A (zh) 编辑rna的方法和组合物
JP2022533673A (ja) プログラム可能塩基エディターシステムを用いた一塩基多型編集法
CN110520163A (zh) 独立于dna双链断裂的靶向基因编辑平台及其用途
US20240167008A1 (en) Novel crispr enzymes, methods, systems and uses thereof
US20230383277A1 (en) Compositions and methods for treating glycogen storage disease type 1a
US20260043013A1 (en) Single base substitution protein, and composition comprising same
EP4692337A1 (en) Cas protein and mutant thereof, and corresponding gene editing system and use thereof
US12576151B2 (en) Fratricide resistant modified immune cells and methods of using the same
US20240254464A1 (en) Cleavage-inactive cas12f1, cleavage-inactive cas12f1-based fusion protein, crispr gene-editing system comprising same, and preparation method and use thereof
US20240301405A1 (en) Guide rnas for crispr/cas editing systems
US20240327813A1 (en) Crispr enzymes, methods, systems and uses thereof
US20250340901A1 (en) Gene editing components, systems, and methods of use
WO2023120658A1 (ja) 酵素、複合体、組換えベクター、遺伝性疾患治療薬及びポリヌクレオチド
CN115244176A (zh) 向导rna-cas蛋白复合物的缀合物
WO2025045078A1 (zh) 一种用于调控rna甲基化修饰的编辑系统及其应用
WO2023049935A1 (en) Compositions and methods for treating hepatitis b virus infection
KR20240141708A (ko) 바이러스 가이드 rna 전달
US9816077B2 (en) Use of integrase for targeted gene expression
US20250288690A1 (en) Rna base editing compositions, systems, methods and uses thereof
Economos Peptide Nucleic Acids and CRISPR-Cas9: Mechanisms and Rational Applications for Gene Editing Systems
KR20250177381A (ko) 징크 핑거 단백질 및 닉카제를 포함하는 신규한 염기 에디터
KR20260051041A (ko) 생체안전성 및 효율성이 개선된 트랜스포사제

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22911360

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2023569543

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 202417052374

Country of ref document: IN

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022911360

Country of ref document: EP

Effective date: 20240724

WWE Wipo information: entry into national phase

Ref document number: 202280092054.8

Country of ref document: CN

WWP Wipo information: published in national office

Ref document number: 18723452

Country of ref document: US