WO2022004765A1 - ゲノム編集用組成物 - Google Patents

ゲノム編集用組成物 Download PDF

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WO2022004765A1
WO2022004765A1 PCT/JP2021/024675 JP2021024675W WO2022004765A1 WO 2022004765 A1 WO2022004765 A1 WO 2022004765A1 JP 2021024675 W JP2021024675 W JP 2021024675W WO 2022004765 A1 WO2022004765 A1 WO 2022004765A1
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sgrna
mrna
micelles
sequence
cas
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French (fr)
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一則 片岡
智士 内田
サエド アムジャド ヨセフ アッバシ
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公益財団法人川崎市産業振興財団
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

  • the present invention relates to a composition for genome editing. It also relates to a polyion complex used in the genome editing composition. Further, the present invention relates to a pharmaceutical composition containing the composition for genome editing.
  • the CRISPR / Cas9 system is a genome editing tool that is expected to be applied to the treatment of a wide range of diseases such as genetic diseases and viral infections.
  • genome editing using the CRISPR / Cas9 system the target region targeted by the guide RNA is double-stranded by the Cas9 nuclease. Double-stranded DNA is known to be repaired by homologous recombinant repair (Homologous Directed Repair) or non-homologous end recombination (Non-Homologous End-Joining Repair: NHEJ).
  • HDR any sequence can be integrated into the target region by introducing the donor DNA into the cell.
  • NHEJ several nucleotides are inserted or deleted to induce gene knockdown.
  • Non-Patent Document 1 mRNA is considered to be more persistent than ribonucleoprotein and have a lower risk of off-targeting than plasmid DNA or viral vectors.
  • Guide RNA is about 100 nucleotides (nt) and is easily degraded as compared with mRNA. Therefore, there is a demand for a technique capable of stably delivering guide RNA together with Cas9.
  • the present invention provides a pharmaceutical composition containing a composition for genome editing capable of stably delivering guide RNA, a polyion complex used for the composition for genome editing, and the composition for genome editing. Make it an issue.
  • a pharmaceutical composition containing a genome editing composition capable of stably delivering guide RNA, a polyion complex used for the genome editing composition, and the genome editing composition.
  • mice co-encapsulated with Cy5-sgRNA and Cy3-Cas9 mRNA and the Cy5-sgRNA micelle (sgRNA micelle) are shown.
  • the results of FCS analysis of co-encapped micelle (co-encap. Micelle) co-encapsulated with Cy5-sgRNA and Cy3-Cas9 mRNA and Cy5-sgRNA micelle (sgRNA micelle) are shown.
  • the results of DLS analysis of co-encapsulated micelles co-encap. Micelle), sgRNA micelles (sgRNA micelles), and Cas9 mRNA micelles (Cas9 mRNA micelles) co-encapsulated with sgRNA and Cas9 mRNA are shown.
  • the results of evaluating the nuclease stability of co-encapsulated micelles co-encapsulated with sgRNA and Cas9 mRNA and mixed micelles mixed with sgRNA micelles and Cas9 mRNA micelles are shown.
  • the results of genome editing by introducing co-encapsulated micelles co-encapsulated with Ai9 sgRNA and Cas9 mRNA into each tissue of Ai9 mouse are shown.
  • the above figure is a diagram illustrating a scheme of genome editing using Ai9 sgRNA in Ai mice.
  • the figure below is a fluorescence micrograph showing the results of genome editing in the lungs, brain, joints, and muscles of Ai mice.
  • Co-encapsulated micelles co-encapsulated with Ai9 sgRNA and Cas9 mRNA or mixed micelles containing Ai9 sgRNA micelles and Cas9 mRNA micelles are introduced into the brain of Ai9 mice to introduce the genome.
  • the result of editing is shown.
  • Ai9 mouse brain is mixed with co-encap.
  • the result of genome editing by introducing the Naked mixture (Naked) was shown.
  • the term "gene” means a polynucleotide containing at least one open reading frame encoding a particular protein.
  • the gene can include both exons and introns.
  • the term "donor DNA” refers to DNA used for repairing double-strand breaks in DNA and is exogenous DNA that can be homologously recombined with DNA around the target region.
  • the donor DNA contains a base sequence adjacent to the target region as a homology arm.
  • the homology arm consisting of the base sequence adjacent to the 5'side of the target region is referred to as "5'homology arm”
  • the homology arm consisting of the base sequence adjacent to the 3'side of the target sequence is referred to as "3'homology arm”. Called.
  • the donor DNA can contain the desired base sequence between the 5'homology arm and the 3'homology arm.
  • the length of each homology arm is preferably 3 kb or more, and is usually about 5 to 10 kb.
  • the lengths of the 5'homology arm and the 3'homology arm may be the same or different, but are preferably the same.
  • Cas protein refers to a CRISPR-associated protein.
  • the Cas protein forms a complex with a guide RNA and exhibits endonuclease activity or nickase activity.
  • the Cas protein is not particularly limited, and examples thereof include Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, and C2c3 protein.
  • Cas proteins include wild-type Cas proteins and their homologs (paralogs and orthologs), as well as variants thereof, as long as they exhibit endonuclease activity or nickase activity in conjunction with guide RNA.
  • the Cas protein may be a dCas protein in which endonuclease activity or nickase activity has been deactivated.
  • the dCas protein works with the guide RNA to bind to the DNA of the target region.
  • a Cas protein exhibiting endonuclease activity, nickase activity, or DNA binding activity in cooperation with a guide RNA may be referred to as "functional Cas protein".
  • the Cas protein is involved in a Class 2 CRISPR / Cas system, more preferably a type II CRISPR / Cas system.
  • a preferred example of the Cas protein is the Cas9 protein.
  • Cas9 protein refers to the Cas protein involved in the type II CRISPR / Cas system.
  • the Cas9 protein forms a complex with the guide RNA and exhibits the activity of collaborating with the guide RNA to cleave the DNA of the target region.
  • Cas9 protein includes wild-type Cas9 protein and its homologs (paralogs and orthologs) and variants thereof, as long as they have the above-mentioned activity.
  • the wild-type Cas9 protein has a RuvC domain and an HNH domain as nuclease domains.
  • the Cas9 protein may be one in which either the RuvC domain or the HNH domain is inactivated.
  • the Cas9 protein may be one in which both the RuvC domain and the HNH domain are inactivated (dCas9 protein).
  • the species from which the Cas9 protein is derived is not particularly limited, and examples thereof include bacteria belonging to the genus Streptococcus, Staphylococcus, Neisseria, and Treponema. More specifically, S. pyogenes, S. streptococcus. thermophilus, S.A. aureus, N. et al. Meningitidis, or T.I. Cas9 protein derived from detentola and the like can be mentioned. In a preferred embodiment, the Cas9 protein is S. It is a Cas9 protein derived from pyogenes.
  • the guide RNA comprises a CRISPR RNA (crRNA) and a transactivated CRISPR RNA (tracrRNA).
  • the crRNA is involved in binding to a target region on the genome, and the tracrRNA is involved in binding to the Cas protein.
  • the crRNA comprises a spacer sequence and a repeat sequence, the spacer sequence binding to the complementary strand of the target sequence in the target region.
  • the tracrRNA comprises an anti-repeat sequence and a 3'tail sequence.
  • the anti-repeat sequence has a sequence complementary to the repeat sequence of crRNA and forms a base pair with the repeat sequence, and the 3'tail sequence usually forms three stem loops.
  • the guide RNA may be a single-stranded guide RNA (sgRNA) in which the 5'end of tracrRNA is linked to the 3'end of crRNA.
  • the guide RNA may be one in which crRNA and tracrRNA are separate RNA molecules and base pairs are formed by repeat sequences and anti-repeat sequences.
  • the guide RNA is sgRNA.
  • the repeat sequence of crRNA and the sequence of tracrRNA can be appropriately selected depending on the type of Cas protein.
  • the repeat sequence of crRNA and the sequence of tracrRNA those derived from the same bacterial species as Cas protein can be used.
  • S the length of sgRNA can be about 50 to 220 nucleotides (nt), preferably about 60 to 180 nt, and more preferably about 80 to 120 nt.
  • the length of crRNA can be about 25 to 70 bases including the spacer sequence, and is preferably about 25 to 50 nt.
  • the length of the tracrRNA can be about 10 to 130 nt, preferably about 30 to 80 nt.
  • the repeat sequence of crRNA may be the same as that in the bacterial species from which the Cas protein is derived, or may be the one in which a part of the 3'end is deleted.
  • the tracrRNA may have the same sequence as the mature tracrRNA in the bacterial species from which the Cas protein is derived, or may be a terminal-cleaving type in which the 5'end and / or the 3'end of the mature tracrRNA is cleaved.
  • the tracrRNA can be a terminal-cleaving type in which about 1 to 40 nucleotide residues are removed from the 3'end of the mature tracrRNA.
  • the tracrRNA can be a terminal-cleaving type in which about 1 to 80 nucleotide residues are removed from the 5'end of the mature tracrRNA. Further, the tracrRNA can be, for example, a terminal-cleaving type in which about 1 to 20 nucleotide residues are removed from the 5'end and about 1 to 40 nucleotide residues are removed from the 3'end.
  • Various crRNA repeat sequences and tracrRNA sequences for sgRNA design have been proposed, and those skilled in the art can design sgRNAs based on known techniques (eg, Jinek et al. (2012) Science, 337, 337, 816-21; Mali et al.
  • target sequence refers to a DNA sequence in the genome that is the target of cleavage or binding by the Cas protein.
  • the target sequence needs to be a sequence adjacent to the 5'side of the protospacer flanking motif (PAM).
  • PAM protospacer flanking motif
  • As the target sequence a sequence of 17 to 30 bases (preferably 18 to 25 bases, more preferably 19 to 22 bases, still more preferably 20 bases) adjacent immediately before the 5'side of PAM is selected.
  • a known design tool such as CRISPR DESIGN (crispr.mit.edu/) can be used to design the target sequence.
  • target region refers to a genomic region containing a target sequence and its complementary strand.
  • the PAM corresponding to the Cas9 protein of aureus is "NNGRRT” or “NNGRR (N)".
  • N The PAM corresponding to the Cas9 protein of meningitidis is "NNNNGATT”.
  • T It is "NAAAAC” corresponding to Cas9 protein of detentola.
  • R indicates A or G
  • N indicates A, T, G or C.
  • spacer sequence and "guide sequence” are used interchangeably and refer to a sequence contained in a guide RNA that can bind to the complementary strand of the target sequence. Normally, the spacer sequence is the same sequence as the target sequence (however, T in the target sequence is U in the spacer sequence).
  • the spacer sequence may contain a mismatch of one or more bases (eg, 2 bases, 3 bases, 4 bases, etc.) to the target sequence as long as it maintains its ability to bind to the complementary strand of the target sequence.
  • silent mutation refers to a gene mutation in which the amino acid sequence of the encoding protein does not change.
  • codon optimization refers to replacing at least one codon in the original base sequence with a codon that is more frequently used in the organism of interest, while preserving the original amino acid sequence.
  • the codon usage frequency table is easily available, for example, in "Codon Usage Database” (www.kazusa.or.jp/codon/) provided by the Kazusa DNA Research Institute. For example, a codon usage frequency table can be used to optimize codons.
  • Computer algorithms for codon-optimizing specific sequences for expression in specific animal species are also known. Computer algorithms for codon optimization are available, for example, in Gene Forge (Aptagen; Jacobus, PA) and the like.
  • micelle refers to a spherical molecular assembly formed by the association of multiple molecules.
  • PIC polyion complex
  • PIC micelle refers to the PIC forming the micelle.
  • cationic polymer block refers to a block with a positive charge in a block copolymer.
  • the cationic polymer block has a cationic monomer unit.
  • the cationic polymer block may have uncharged monomer units in addition to the cationic monomer units as long as the entire block has a positive charge.
  • polyamino acid refers to a polymer obtained by polymerizing an amino acid and a polymer obtained by chemically modifying the polymer.
  • the amino acid used for the polymerization may be a natural amino acid or an unnatural amino acid.
  • the amino acid used for the polymerization may be one kind or two or more kinds.
  • amino acid monomer unit refers to a monomer unit derived from an amino acid or a monomer unit obtained by chemically modifying the monomer unit in a polyamino acid.
  • hydrophilic polymer block refers to a block that exhibits hydrophilicity in a block copolymer.
  • the hydrophilic polymer block has a hydrophilic monomer unit.
  • the hydrophilic polymer block may have a hydrophobic monomer unit in addition to the hydrophilic monomer unit as long as the entire block is hydrophilic.
  • the hydrophilicity / hydrophobicity of the compound can be defined, for example, by the logP value.
  • the logP value is a logarithmic value of the octanol / water partition coefficient ( Power ) and is an effective parameter capable of characterizing the hydrophilicity / hydrophobicity of a wide range of compounds. When the logP value increases on the plus side across 0, it means that the hydrophobicity increases, and when it increases on the minus side, it means that the hydrophilicity increases.
  • the hydrophilic polymer block may have a negative LogP value.
  • polymerization degree refers to the number of monomer units in a polymer. "Average degree of polymerization” means a number average degree of polymerization unless otherwise specified.
  • the numerical value of the molecular weight with the unit "Da" is the molar mass.
  • the term “comprise” means that components other than the target component may be included.
  • the term “consist of” means that it does not include any component other than the target component.
  • the term “essentially consist of” does not include components other than the target component in a mode that exerts a special function (such as a mode in which the effect of the invention is completely lost). means.
  • the term “comprise” includes the “consist of” mode and the “essentially consist of” mode.
  • Proteins, peptides, polynucleotides (DNA, RNA), vectors, and cells can be isolated. "Isolated” means a state isolated from the natural state.
  • the proteins, peptides, polynucleotides (DNA, RNA), vectors, and cells described herein are isolated proteins, isolated peptides, isolated polynucleotides (isolated DNA,). It can be an isolated RNA), an isolated vector, and an isolated cell.
  • the invention provides a polyion complex comprising a block copolymer having a cationic polymer block and a hydrophilic polymer block, mRNA encoding a Cas protein, and guide RNA.
  • P is a block copolymer having a cationic polymer block (b1) and a hydrophilic polymer block (b2) (hereinafter, also referred to as “block copolymer (P)”).
  • Reference numeral 11 is an mRNA encoding a Cas protein (hereinafter, also referred to as “Cas mRNA”).
  • Reference numeral 12 is a guide RNA (sgRNA) having an arbitrary spacer sequence.
  • the block copolymer (P) has a cationic polymer block (b1).
  • RNA on the other hand, is an anionic polymer. Therefore, when the block copolymer (P), Cas mRNA (11), and guide RNA (12) are mixed in an aqueous solution, Cas mRNA (11) and guide RNA (12) are ionized in the cationic polymer block (b1). Combine to form a PIC.
  • the PIC is preferably a PIC micelle (1).
  • the hydrophilic polymer block (b2) is located on the outside and the cationic polymer block (b1) is located on the inside.
  • FIG. 1 shows a single-stranded guide RNA (sgRNA) as a guide RNA (12).
  • sgRNA single-stranded guide RNA
  • stable micelles cannot be formed even when mixed with the block copolymer (P).
  • stable PIC micelles (1) can be formed by mixing guide RNA (12) with block copolymer (P) together with Cas mRNA (11) having a longer chain length.
  • the block copolymer (P) has a cationic polymer block (b1) and a hydrophilic polymer block (b2).
  • the cationic polymer block (b1) is not particularly limited and can be selected according to the application of the PIC. When applying PIC to a living body, it is preferable to use a highly biocompatible material for the cationic polymer block (b1).
  • Examples of the cationic polymer block (b1) include a cationic polyamino acid block.
  • the cationic polyamino acid block has a cationic amino acid monomer unit and has a cationic property as a whole.
  • Examples of the cationic amino acid monomer unit include a cationic natural amino acid monomer unit derived from a natural amino acid having a cationic side chain; and a cationic unnatural amino acid monomer derived from an unnatural amino acid having a cationic side chain. Units; and cationic unnatural amino acid monomer units obtained by chemical modification of natural amino acids or unnatural amino acids.
  • Examples of the cationic natural amino acid monomer unit include amino acid monomer units derived from cationic natural amino acids such as histidine, tryptophan, ornithine, arginine, and lysine.
  • the cationic natural amino acid is preferably arginine, ornithine or lysine, more preferably ornithine or lysine, and even more preferably lysine.
  • An amino acid monomer unit (hereinafter, also referred to as “unnatural amino acid monomer unit (c1)”) having (an integer of) as a side chain can be mentioned.
  • p is preferably 1 to 4, more preferably 1 to 3, and even more preferably 2 or 3.
  • Examples of R a, - (CH 2) q -CO- (q is an integer of from 0 to 5) is preferred.
  • q 0 to 3 is more preferable, 0 to 2 is further preferable, and 0 or 1 is particularly preferable.
  • Unnatural amino acids monomer units (c1) for example, an amino acid monomer units derived from L- aspartic acid 4-benzyl or L- glutamic acid 5-benzyl, NH 2 - (CH 2 -CH 2 -NH) r- Examples thereof include those obtained by reacting an amine compound represented by H (r is an integer of 1 to 5). Examples of the amine compound include ethylenediamine (EDA), diethylenetriamine (DET), triethylenetetramine (TET), tetraethylenepentamine (TEP) and the like.
  • EDA ethylenediamine
  • DET diethylenetriamine
  • TET triethylenetetramine
  • TEP tetraethylenepentamine
  • Examples of the unnatural amino acid monomer unit (c1) include amino acid monomer units represented by the following general formula (c1-1).
  • R a represents a divalent linking group
  • R b represents a single bond, a methylene group or an ethylene group
  • p is an integer of 1-5.
  • the cationic polyamino acid block may be composed of a cationic natural amino acid monomer unit or a cationic unnatural amino acid monomer unit, and may be a cationic natural amino acid monomer unit and a cationic unnatural amino acid. It may consist of a monomer unit.
  • the backbone of the cationic polyamino acid block is formed by peptide bonds.
  • the cationic polyamino acid block preferably has an unnatural amino acid monomer unit (c1).
  • the amino acid monomer unit represented by the general formula (c1-1) is preferable, and it is represented by any one of the formulas (c1-1-1) to (c1-1-6).
  • the amino acid monomer unit to be used is more preferable, and the amino acid monomer unit represented by the formula (c1-1-1) or the formula (c1-1-4), or the formula (c1-1-2) or the formula (c1-1-1).
  • the amino acid monomer unit represented by 5) is more preferable.
  • the amino acid monomer unit represented by the formula (c1-1-1) and the formula (c1-1-4), the formula (c-1-1-2) and the formula (c1-1-5) may be used.
  • the amino acid monomer unit represented or the amino acid monomer unit represented by the formulas (c1-1-3) and (c1-1-6) is mixed in the cationic polyamino acid block.
  • the cationic polyamino acid block is particularly preferably one having an amino acid monomer unit represented by the formula (c1-1-1) and an amino acid monomer unit represented by the formula (c1-1-4).
  • the ratio of the unnatural amino acid monomer unit (c1) to all the monomer units in the cationic polymer block (b1) is not particularly limited, but is preferably 40% or more, for example.
  • Examples of the ratio of the unnatural amino acid monomer unit (c1) include 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more.
  • the proportion of the unnatural amino acid monomer unit (c1) may be 100%.
  • PAsp (DET) can be obtained by reacting a polymer of 4-benzyl L-aspartate with diethylenetriamine (DET), and has an amino acid monomer unit represented by the above formula (c1-1-1) and a formula. It has an amino acid monomer unit represented by (c1-1-4).
  • PAsp (TET) can be obtained by reacting a polymer of 4-benzyl L-aspartate with triethylenetetramine (TET), and the amino acid monomer unit represented by the above formula (c1-1-2) and It has an amino acid monomer unit represented by the formula (c1-1-5).
  • PAsp can be obtained by reacting a polymer of 4-benzyl L-aspartate with tetraethylenepentamine (TEP), and is an amino acid monomer unit represented by the above formula (c1-1-3). And has an amino acid monomer unit represented by the formula (c1-1-6).
  • the average degree of polymerization of the cationic polymer block (b1) is not particularly limited, and examples thereof include 15 or more, 20 or more, 30 or more, 40 or more, or 50 or more.
  • the upper limit of the average degree of polymerization of the cationic polymer block is not particularly limited, and examples thereof include 500 or less, 300 or less, 200 or less, 150 or less, 120 or less, or 100 or less.
  • Examples of the average degree of polymerization of the cationic polymer block (b1) include 15 to 500, 20 to 300, 30 to 150, 40 to 120, and 50 to 100.
  • the average degree of polymerization can be calculated from the NMR spectrum.
  • the number average molecular weight of the cationic polymer block (b1) is not particularly limited, and examples thereof include 4,000 or more, 5,000 or more, 8,000 or more, 10,000 or more, or 12,000 or more.
  • the upper limit of the number average molecular weight of the cationic polymer block (b1) is not particularly limited, and examples thereof include 150,000 or less, 100,000 or less, 80,000 or less, 50,000 or less, or 30,000 or less. ..
  • the hydrophilic polymer block (b2) is not particularly limited and can be selected according to the application of the PIC. When applying PIC to a living body, it is preferable to use a highly biocompatible material for the hydrophilic polymer block (b2).
  • the hydrophilic polymer block preferably has no charge as a whole block.
  • a polyalkylene glycol block or a poly (2-oxazoline) block is preferable, and a polyalkylene glycol block is more preferable.
  • a polyethylene glycol (PEG) block is particularly preferable.
  • the hydrophilic polymer block (b2) may be a linear polymer block or a branched chain polymer block. From the viewpoint of increasing the bulk of the hydrophilic polymer block (b2), it is preferable to use a branched chain polymer block.
  • the hydrophilic polymer block (b2) is a branched chain polymer block, it may have one branch and a plurality of polymer chains may extend from the branch.
  • the number average molecular weight of the hydrophilic polymer block (b2) is not particularly limited, but is, for example, 5,000 or more, 10,000 or more, 12,000 or more, 15,000 or more, 20,000 or more, 30,000 or more, and the like. Or 40,000 or more can be mentioned.
  • the upper limit of the number average molecular weight is not particularly limited, and examples thereof include 80,000 or less, 70,000 or less, 60,000 or less, or 50,000 or less.
  • the number average molecular weight of the hydrophilic polymer block (b2) is, for example, 5,000 to 80,000, 10,000 to 70,000, 10,000 to 50,000, or 12,000 to 50,000. Can be mentioned.
  • each branched chain may have the above number average molecular weight.
  • the block copolymer (P) preferably has one cationic polymer block (b1) and one hydrophilic polymer block (b2). It is preferable that the hydrophilic polymer block (b2) is bonded to one end of the cationic polymer block (b1). Since the block copolymer (P) has one cationic polymer block (b1) and one hydrophilic polymer block (b2), respectively, PIC micelles are easily formed when mixed with Cas mRNA and guide RNA.
  • PEG-PAsp EDA
  • PEG-PAsp DET
  • PEG-PAsp TAT
  • PEG-PAsp TCP
  • PEG-PLys PEG-PArg
  • PEG-POR PEG-PAsp (DET) and PEG-PLys are more preferred.
  • mRNA ⁇ MRNA encoding Cas protein (Cas mRNA)>
  • the mRNA is not particularly limited as long as it encodes a Cas protein.
  • the Cas protein is preferably the Cas9 protein, and S.I. Cas9 protein derived from pyogenes is more preferred.
  • mRNA means messenger RNA and usually contains a 5'untranslated region (5'UTR), a coding region and a 3'untranslated region (3'UTR).
  • the mRNA further usually comprises a 5'end cap structure (5'Cap) and a 3'end poly A sequence.
  • Cas mRNA examples include the following. (1) An mRNA containing 5'Cap, 5'UTR, Cas protein coding region, 3'UTR, and polyA in this order. (2) An mRNA containing 5'Cap, 5'UTR, Cas protein coding region, and polyA in this order. (3) An mRNA containing 5'UTR, Cas protein coding region, 3'UTR, and polyA in this order. (4) mRNA containing 5'UTR, Cas protein coding region, and polyA in this order. (5) An mRNA containing 5'Cap, 5'UTR, Cas protein coding region, and 3'UTR in this order.
  • mRNA containing 5'Cap, 5'UTR, and Cas protein coding region in this order (6) mRNA containing 5'Cap, 5'UTR, and Cas protein coding region in this order. (7) An mRNA containing 5'UTR, Cas protein coding region, and 3'UTR in this order. (8) mRNA containing 5'UTR and Cas protein coding region in this order.
  • the Cas protein coding region is a region having a base sequence encoding a Cas protein (hereinafter, also referred to as "Cas protein coding sequence").
  • the Cas protein coding sequence may be identical to the coding sequence in the native Cas protein mRNA.
  • the Cas protein coding sequence may have a mutation in the native coding sequence as long as it encodes a functional Cas protein.
  • the Cas protein coding sequence may have a silent mutation in the natural coding sequence. Further, it may be codon-optimized according to the species to be applied. Further, in order to stabilize the mRNA, the G / C content may be increased.
  • Cas mRNA can be produced by a known method. For example, it can be produced by transcribing the template DNA encoding the Cas protein in vitro. Cas mRNA can be prepared, for example, according to the method described in Blood 108 (13) (2006) 4009-17. Specifically, the template DNA in which the poly A / T chain is integrated downstream of the Cas protein coding sequence is cleaved immediately downstream of the poly A / T chain, and a transcription enzyme (RNA polymerase) and a nucleotide (ATP, GTP, CTP) are cleaved. , UTP), in vitro transcription in a buffer solution containing 5'cap analog, followed by purification of mRNA.
  • RNA polymerase RNA polymerase
  • ATP a transcription enzyme
  • GTP GTP
  • CTP nucleotide
  • the length of the polyA sequence of Cas mRNA is, for example, 10 to 500 bases, preferably 30 to 300 bases, and more preferably 60 to 250 bases.
  • the 5'UTR and 3'UTR sequences of Cas mRNA may have 100% sequence identity with the naturally occurring 5'UTR and 3'UTR sequences of Cas mRNA, with some bases substituted. It may be a thing.
  • the 5'UTR sequence of Cas mRNA may be partially or wholly replaced with the 5'UTR sequence of another gene.
  • the 3'UTR sequence of Cas mRNA may be partially or wholly replaced with the 3'UTR sequence of another gene.
  • Examples of the 5'UTR sequence and 3'UTR sequence of other genes include globin, hydroxysteroid (17- ⁇ ) dehydrogenase 4, or 5'UTR sequence and 3'UTR sequence of mRNA encoding albumin.
  • Cas mRNA may be chemically modified with some or all of the nucleotide residues. Chemical modifications of nucleotide residues may be made to any of the ribose, base, and phosphodiester bonds. If the nucleotide residue of Cas mRNA is not chemically modified, it can be expected that the translation process will be hardly impaired because the mRNA is natural. Further, when chemically modifying the nucleotide residue of Cas mRNA, improvement of mRNA resistance to degrading enzyme and reduction of immunogenicity can be expected.
  • Examples of the chemically modified base that the nucleotide residue of Cas mRNA can have include a methylated base (for example, 5-methylcytosine), a sulfur-modified base (for example, 2-thiouridine), a pseudouridine, and an N1 methylpseudouridine, 5 Examples thereof include, but are not limited to, methoxyuridine.
  • Cas mRNA may or may not have 5'Cap. It is expected that Cas mRNA will be stabilized by having 5'Cap.
  • the 5'Cap usually has a structure in which 7-methylguanosine (m 7 G) is bound to the 5'end of the 5'UTR by a 5'-5'binding.
  • Cas mRNAg may be a methoxy group by methylating the hydroxyl group at the 2'position of ribose at the 1st and / or 2nd nucleotide residues at the 5'end. It is known that the modification using the methoxy group improves the expression efficiency.
  • Cas mRNA may be used.
  • Examples of commercially available Cas mRNA include CleanCap (registered trademark) Cas9 mRNA and CleanCapl Cas9 Nickase mRNA (both manufactured by Trilink).
  • the target sequence targeted by the guide RNA is not particularly limited and can be appropriately designed according to the purpose of genome editing.
  • the target of genome editing can be any cell.
  • the target of genome editing may be a prokaryote or a eukaryote. Since it can be delivered to any site in the living body while suppressing the degradation of guide RNA, it is preferably applied to eukaryotes, and more preferably to multicellular organisms.
  • organisms targeted for genome editing include mammals such as humans, monkeys, mice, rats, dogs, cats, rabbits, cows, horses, pigs, goats, and sheep; birds such as chickens; snakes, lizards, etc.
  • the target sequence can be designed by a known method based on the base sequence of the target region on the genome.
  • the target sequence can be 17 to 30 bases adjacent to the 5'side of PAM present in the target region on the genome.
  • the base sequence of the target region may be obtained from various databases such as GenBank and UniProt, for example.
  • the guide RNA may have a chemically modified nucleotide residue. Having a chemically modified nucleotide residue can be expected to improve the enzyme resistance of the guide RNA.
  • Chemical modifications of nucleotide residues may be made to any of the ribose, base, and phosphodiester bonds. Examples of the chemical modification include modification of methylating the 2'-hydroxyl group of ribose, modification of substituting the 2'-hydroxyl group of ribose with a fluorine atom, and modification of a phosphodiester bond as a phosphothioate bond. Not limited.
  • Chemically modified nucleotide residues include 2'-O-methyl-nucleotide residues, 2'-O-methyl 3'phosphorothioate nucleotide residues, and 2'-deoxy-2'-fluoro, in addition to those listed above. -Includes, but is not limited to, nucleotide residues and the like.
  • the position of the chemically modified nucleotide residue is not particularly limited, and examples thereof include the 5'end and the 3'end of sgRNA.
  • the chemically modified nucleotide residue is preferably introduced at a position that does not interact with the Cas protein.
  • the ratio of Cas mRNA and guide RNA to the block copolymer (P) is not particularly limited.
  • the ratio of Cas mRNA and guide RNA to the block copolymer (P) can be adjusted based on, for example, the ratio of charges assumed when the pH is 7.4.
  • the charge ratio (+/-) of the block copolymer (P) (+) to Cas mRNA (-) and guide RNA (-) can be, for example, 1 to 20, preferably 1 to 8. ⁇ 2 is more preferable. Specific examples of the charge ratio include 1.5.
  • the donor DNA contains a base sequence adjacent to the target region as a homology arm.
  • the donor vector can contain a desired base sequence (hereinafter, may be referred to as “knock-in sequence”) between the 5'homology arm and the 3'homology arm.
  • the knock-in sequence is not particularly limited and may be any sequence.
  • the knock-in sequence may be, for example, a sequence for gene knockout, a sequence for base substitution, or an arbitrary gene sequence.
  • the knock-in sequence may be a sequence that corrects an abnormal base of the disease-causing gene.
  • the donor DNA may be a circular DNA vector (for example, a plasmid vector) or a linear DNA vector.
  • the donor DNA may contain other sequences in addition to the homology arm and knock-in sequences. Examples of other sequences include marker genes (antibiotics resistance genes, fluorescent protein genes, auxotrophic genes, etc.), replication initiation sites, genes encoding proteins that bind to replication initiation sites and control replication, and the like. Be done.
  • the PIC of this embodiment can form stable PIC micelles by containing block copolymer (P), Cas mRNA, and guide RNA.
  • block copolymer (P) As shown in Examples described later, when guide RNA is mixed with block copolymer (P) to form PIC, stable PIC micelles cannot be formed and micelles tend to disintegrate. Therefore, it is susceptible to the action of enzymatic decomposition in the living body. Even when the guide RNA is encapsulated in a known carrier such as a liposome, it is difficult to stably deliver the guide RNA to the target site in vivo.
  • stable PIC micelles can be formed by preparing a PIC containing a block copolymer (P), Cas mRNA, and a guide RNA.
  • P block copolymer
  • Cas mRNA and guide RNA are stably retained in the PIC micelle, and actions such as enzymatic degradation can be suppressed. Therefore, even when administered to a living body, these RNAs can be stably delivered to a target site.
  • the PIC is preferably a PIC micelle.
  • PIC micelles since the hydrophilic polymer block (b2) is present in the outer shell of the micelle, it is easily taken up by cells by physicochemical endocytosis. PIC micelles taken up by cells release Cas mRNA and guide RNA. Cas mRNA is translated intracellularly to produce Cas protein. The Cas protein is guided to the target region by the guide RNA and cleaves the target region.
  • the PIC contains donor DNA
  • the double-strand cut target region can be repaired by HDR.
  • HDR the DNA in the target region is replaced with the knock-in sequence of the donor DNA.
  • the double-strand cut target region is repaired by NHEJ. This leads to the insertion or deletion of several nucleotides, leading to knockdown of genes present in the target region.
  • the composition for genome editing of the present embodiment may contain an arbitrary component in addition to the PIC as long as the effect of the present invention is not impaired.
  • the optional component may be donor DNA.
  • the donor DNA may be in a naked state, or may be encapsulated or bound to a carrier or the like.
  • the carrier a known carrier for drug delivery can be used.
  • examples of the carrier include a cationic polymer and a lipid nucleic acid carrier.
  • the cationic polymer include the block copolymer (P).
  • Lipid nucleic acid carriers include those formed by lipids or cationic lipids.
  • DOTMA N, N-trimethylammonium chloride
  • 2,3-dioreyloxy-N- [2- (spermine carboxylamide) 2,3-dioreyloxy-N- [2- (spermine carboxylamide)
  • composition for genome editing of this embodiment contains the PIC, Cas mRNA and guide RNA can be stably delivered into the target cells. Therefore, genome editing can be performed efficiently.
  • the invention provides a pharmaceutical composition comprising said genome editing composition.
  • the genome editing composition can be used as a pharmaceutical composition for treating the disease.
  • the target of application of the pharmaceutical composition of this embodiment has a disease-causing gene targeted by guide RNA.
  • diseases to be treated of the pharmaceutical composition of the present embodiment include Huntington's disease, vulnerability X syndrome, pulmonary fibrosis, asthma, muscular dystrophy, lower limb ischemia, rheumatoid arthritis, knee osteoarthritis and the like. However, it is not limited to these.
  • the administration route of the pharmaceutical composition of this embodiment is not particularly limited.
  • the routes of administration include, for example, intravenous administration, intraarterial administration, oral administration, intratissue administration (for example, intravesical administration, intrathoracic administration, intraperitoneal administration, intraocular administration, intracerebral administration), transdermal administration, and transfusion. Mucosal administration, transpulmonary administration, transrectal administration and the like can be mentioned.
  • the pharmaceutical composition of the embodiment is administered in the form of a dosage form suitable for these administrations, for example, various injections, oral preparations, infusions, inhalants, eye drops, ointments, lotions, or suppositories. To.
  • the route of administration can be appropriately selected according to the disease-causing gene targeted by the guide RNA.
  • the pharmaceutical composition of the present embodiment can be locally administered to a lesion of a disease caused by a disease-causing gene.
  • intracerebral administration can be selected in the case of diseases that cause abnormalities in the cranial nerves such as Huntington's disease and Fragile X syndrome.
  • intracerebral administration can be selected in the case of pulmonary fibrosis and diseases that cause abnormalities in the lungs such as asthma.
  • intrapulmonary administration can be selected.
  • intramuscular administration can be selected.
  • intra-articular administration can be selected.
  • the pharmaceutical composition of the present embodiment may be a single dose or a plurality of doses.
  • the administration period and interval can be appropriately selected depending on the type and condition of the disease, the administration route, the age, body weight, sex, etc. of the administration target. Examples of the administration interval include, but are not limited to, 1 to 3 times a day, once every 2 to 3 days, 1 to 3 times a week, and once every 10 days.
  • the dose, administration period, administration interval, etc. of the pharmaceutical composition of the present embodiment can be appropriately selected depending on the type of drug, the type and condition of the disease, the administration route, the age, body weight, sex, etc. of the administration target.
  • the dose of the pharmaceutical composition of the present embodiment can be, for example, a therapeutically effective amount of the PIC.
  • “Therapeutically effective amount” means the amount of active ingredient effective for the treatment or prevention of a disease.
  • the active ingredient is the PIC.
  • the dose of the PIC per administration may be about 0.01 to 1000 mg, about 0.1 to 100 mg, about 0.5 to 50 mg, or about 1 to 10 mg per 1 kg of body weight.
  • the pharmaceutical composition of the present embodiment contains the above-mentioned genome editing composition, Cas mRNA and guide RNA can be stably delivered into cells at a diseased site. Therefore, it is possible to efficiently edit the genome of the disease-causing gene.
  • the invention comprises the step of introducing into cells a polyion complex comprising a block copolymer having a cationic polymer block and a hydrophilic polymer block, mRNA encoding Cas protein, and guide RNA.
  • a polyion complex comprising a block copolymer having a cationic polymer block and a hydrophilic polymer block, mRNA encoding Cas protein, and guide RNA.
  • the invention comprises a polyion complex comprising a block copolymer having a cationic polymer block and a hydrophilic polymer block, an mRNA encoding a Cas protein, and a guide RNA targeting a disease-causing gene.
  • a method for treating the disease which comprises a step of administering the gene to a subject having the gene.
  • the present invention relates to a block copolymer having a cationic polymer block and a hydrophilic polymer block, mRNA encoding a Cas protein, and a disease cause of the disease in the production of a pharmaceutical composition for treating a disease.
  • a polyion complex containing a guide RNA that targets a gene is provided.
  • the invention is a block copolymer having a cationic polymer block and a hydrophilic polymer block for treating a disease, mRNA encoding a Cas protein, and a guide targeting the disease-causing gene of the disease. It provides a polyion complex containing RNA.
  • the present invention targets block copolymers having a cationic polymer block and a hydrophilic polymer block, mRNA encoding Cas protein, and the disease-causing gene of the disease for treating the disease. It provides the use of polyion complexes containing RNA.
  • the polyion complex in each of the above embodiments forms micelles.
  • Preferred examples of the polyion complex are the same as above.
  • Example 1 [Materials and methods] 1.
  • Material CleanCap® Cas9 mRNA was purchased from Trilink (San Diego, CA, USA).
  • Ai9 sgRNA chemically modified with 2'-O-methyl-3'phosphorothioate and anti-Iba1 (rabbit) antibody were purchased from Fujifilm Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
  • Anti-glial fibrous protein and anti-NeuN (rabbit polyclonal) antibody were purchased from Merck KgaA (Darmstadt, Germany).
  • Goat anti-rabbit IgG AlexaFluor 488) was purchased from Thermo Fisher Scientific (Waltham, MA, USA).
  • the plasmid was amplified with Escherichia coli DH5 ⁇ competent cell (Takara Bio Inc., Otsu, Japan), extracted and purified using the Qiagen kit (Hilden, Germany).
  • IVT in-vitro transcription
  • the plasmid was first cleaved using the Dr1 enzyme (New England BioLabs, Ipswich, MA, USA), and the obtained fragment was subjected to gel electrophoresis (0.9% agarose gel, 100V, 100V). 30 minutes).
  • the DNA fragment corresponding to Ai9 sgRNA was extracted using a gel extraction kit (Qiagen, Hilden, Germany).
  • Block copolymers composed of PEG (12kD) and PAsp (DET), or PAsp (DET) homopolymers were synthesized as previously reported (K. Itaka, T. Ishii, Y. Hasegawa, K. Kataoka, Biodegradable polyamino acid-based polycations as safe and effective gene carrier minimizing cumulative toxicity, Biomaterials 31 (13) (2010) 3707-3714.).
  • FCS analysis was performed with the ConfoCor3 module and C-Apochromat 40x, N. et al. A. This was done using a combination system consisting of an LSM 510 with a 1.2 water immersion objective (Carl Zeiss, Oberkochen, Germany). A He-Ne laser (wavelength: 633 nm) was used to excite the Cy5 dye bound to the IVT sgRNA.
  • PIC micelles were prepared from PEG-PAsp (DET) and Cy5-sgRNA or Cy5-sgRNA mixed in a 1: 1 mass ratio and unlabeled Cas9 mRNA.
  • the micelles were diluted with phosphate buffered saline (PBS) and equilibrated for 5 minutes. Each sample was then placed in an 8-well Laboratory-Tek chamber (Nalgene Nunc International, Rochester, NY, USA). The FCS measurements for each sample were repeated 10 times, the autocorrelation curves obtained were averaged and converted to diffusion time using ConfoCor3 software.
  • PBS phosphate buffered saline
  • RT-PCR 7500 FastCaster App And using the primer pair for Ai9 sgRNA, qRT-PCR analysis was performed (Forward: TGGTTTTAGAGCATGAAATAGGCAAG (SEQ ID NO: 2); Reverse: CGGTGCCACTTTTTCAAGTT (SEQ ID NO: 3)).
  • Topical administration For intramuscular administration, 150 ⁇ L of co-encapsulated micelles (16.7 ⁇ g / mL chemically modified sgRNA and 16.7 ⁇ g / mL Cas9 mRNA) were injected into the thigh muscles of Ai9 mice. Mice were anesthetized and a small surgical incision was made in the thigh to facilitate accurate injection and surgically sutured after injection. Mice were allowed to survive for 2 days and then muscle was harvested for observation of tissue sections for genome editing.
  • co-encapsulated micelles (16.7 ⁇ g / mL chemically modified sgRNA and 16.7 ⁇ g / mL Cas9 mRNA
  • the mouse trachea was incised under deep anesthesia, and 50 ⁇ L of co-encapsulated micelle (100 ⁇ g / mL chemically modified sgRNA and 100 ⁇ g / mL Cas9 mRNA) was sprayed into the lung using a microspray. The wound was then surgically sutured. Mice were allowed to survive for 3 days and then muscle was harvested for cross-sectional observation of genome editing. Lungs were harvested for observation of tissue sections for genome editing.
  • mice were anesthetized and a small hole was made at the location of the skull corresponding to the target brain region.
  • 4 ⁇ L of each formulation 100 ⁇ g / mL chemically modified sgRNA and 100 ⁇ g / mL Cas9 mRNA
  • a needle was inserted at a depth of 3 mm and 0.5 mm behind the bregma.
  • the solution was automatically delivered at 1 ⁇ L / min and then the wound was surgically sutured. After the mice were allowed to survive for 3 days, the brain was excised and the sectioning observation of genome editing was performed.
  • mice For intra-articular injection, 20 ⁇ L of co-encapsulated micelles (16.7 ⁇ g / mL chemically modified sgRNA and 16.7 ⁇ g / mL Cas9 mRNA) were injected into the knee joint of Ai9 mice. After the mice were allowed to survive for 4 days, the legs of the mice were collected and the sectioning observation of genome editing was performed.
  • co-encapsulated micelles (16.7 ⁇ g / mL chemically modified sgRNA and 16.7 ⁇ g / mL Cas9 mRNA
  • mice Prior to extraction of target tissue, mice were perfused through the left ventricle of the heart with 10 mL of PBS containing PBS followed by 4% paraformaldehyde (PFA). The extracted tissue was immersed in 4% PFA and kept at 4 ° C. overnight. The next day, the tissue was transferred to PBS containing 10% sucrose and held for 4 hours, followed by transfer to PBS containing 15% sucrose and held at 4 ° C. for an additional 4 hours. Finally, the tissue was transferred to PBS containing 20% sucrose and kept at 4 ° C. overnight.
  • PFA paraformaldehyde
  • Brain, muscle, or lung tissue was embedded in an OCT compound (SciGen Ltd., Singapore) and frozen at -100 ° C. using a cold hexane-pentane mixture.
  • OCT compound SciGen Ltd., Singapore
  • a cryostat Leica Biosystems, Wetzlar, Germany
  • the brain was sectioned at 14 ⁇ m and the lungs and muscles were sectioned at 10 ⁇ m and placed on a sticky glass slide.
  • Tissue sections were dried on glass slides and the OCT compound was rinsed with PBS (repeated 3 times).
  • a cover slide was attached and enclosed on a slide glass using ProLong Gold Antifade Mountain with DAPI (Thermo Fisher Scientific, Waltham, MA, USA). Documents (2.
  • FIG. 2 shows the measurement results of the fluorescence intensity of Cy5-sgRNA micelles and Cy3-Cas9 mRNA micelles. After excitation at 520 nm, which is the excitation wavelength of Cy3, the fluorescence spectrum was measured. Cy3-Cas9 mRNA was confirmed to fluoresce at 570 nm, which is the fluorescence wavelength of Cy3.
  • FIG. 3 shows a mixed micelle (mixed micelles) in which a co-encapsulated micelle (co-encap. Micelle) co-encapsulated with Cy5-sgRNA and Cy3-Cas9 mRNA and a Cy5-sgRNA micelle and Cy3-Cas9 mRNA micelle were mixed.
  • the result of FRET analysis is shown. After excitation at 520 nm, which is the excitation wavelength of Cy3, the fluorescence spectrum was measured. In each case, in addition to the fluorescence of 520 nm, which is the fluorescence wavelength of Cy3, fluorescence was confirmed in the vicinity of 670 nm, which is the fluorescence wavelength of Cy5.
  • the release of sgRNA was confirmed at any dilution rate, and it was particularly remarkable at the 5-fold dilution or more. This result indicates that the co-emulsified micelles stably retain sgRNA in the micelles.
  • FIG. 5 shows the results of FCS analysis of co-encapsulated micelles (co-encap. Micelle) co-encapsulated with Cy5-sgRNA and Cy3-Cas9 mRNA and Cy5-sgRNA micelles (sg RNA micelle). Each micelle was diluted with PBS and equilibrated for 5 minutes. After that, FCS analysis was performed. As shown in FIG. 5, the release of sgRNA was gradual in the co-emulsified micelles. on the other hand. In the sgRNA micelle, sgRNA was released rapidly.
  • FIG. 6 shows co-encapsulated micelles co-encapsulated with sgRNA and Cas9 mRNA, sgRNA micelles, and Cas9 mRNA micelles. ) Is shown as a result of DLS analysis. As shown in FIG. 6, the co-emulsified micelle and Cas9 mRNA micelle had higher uniformity than the sgRNA micelle, and the average particle size was controlled to 100 nm or less.
  • Nuclease stability Figure 7 assesses the nuclease stability of co-encapped micelles co-encapsulated with sgRNA and Cas9 mRNA and mixed micelles with sgRNA micelles and Cas9 mRNA micelles. The result is shown. Co-emulsified micelles or mixed micelles were incubated in 50% FBS at 37 ° C. for 30 minutes. Then, the amount of sgRNA remaining in the micelle was measured by qRT-PCR analysis. As shown in FIG. 7, the co-emulsified micelles had a much higher residual amount of sgRNA than the mixed micelles. This result indicates that the nuclease stability of sgRNA is greatly improved in the co-emulsified micelle.
  • FIG. 8 shows the results of genome editing by introducing co-encapsulated micelles co-encapsulated with Ai9 sgRNA and Cas9 mRNA into each tissue of Ai9 mice.
  • Ai9 mice when genome editing is performed using Ai9 sgRNA, the tdTomato gene is expressed. Therefore, the success of genome editing can be confirmed by detecting the fluorescence of tdTomato (see the upper figure of FIG. 8).
  • FIG. 8 The lower figure is a fluorescence micrograph showing the results of genome editing in the lungs, brain, joints, and muscles. As shown in the lower figure of FIG. 8, the fluorescence of tdTomato could be confirmed in any of the tissues. From this result, it was confirmed that the genome can be edited in vivo by introducing co-emulsified micelles.
  • FIG. 9 is a time-lapse image of a brain section. As shown in FIG. 9, fluorescence of tdTomato was detected in the co-emulsified micelle. On the other hand, no fluorescence of tdTomato was detected in the mixed micelles.
  • a co-encapped micelle co-encap.micelle
  • a PAsp PAsp
  • Homo 93 homopolymer mixture of Ai9 sgRNA and Cas9 mRNA
  • An Ai9 sgRNA were added to the brain of an Ai9 mouse.
  • the results of genome editing by introducing a Naked mixture (naked RNA) mixed with Cas9 mRNA are shown.
  • fluorescence of tdTomato was detected in the co-emulsified micelle.
  • no fluorescence of tdTomato was detected in the homopolymer mixture or the Naked mixture.
  • FIG. 11 shows the results of genome editing by introducing co-encapsulated micelles into the brain of Ai9 mice.
  • FIG. 11 is an image processed by 3D imaging.
  • (A) and (b) are views of 3D images from different angles.
  • the fluorescence of tdTomato was confirmed in the range of about 100 ⁇ m. From this result, it was confirmed that the genome was edited by the introduction of co-emulsified micelles. On the other hand, when a homopolymer mixture, an Naked mixture, or a mixed micelle was introduced into Ai9 mice, fluorescence of tdTomato could not be confirmed (not shown).
  • Example 2 [Materials and methods] 1.
  • PIC micelle 100 ⁇ g / mL sgRNA (10 mM phosphate buffer, pH 7.4) and 100 ⁇ g / mL of polymer (PEG-PLys) dissolved in 10 mM phosphate buffer (pH 7.4) under vortex mixing.
  • Cas9 RNA (10 mM phosphate buffer, pH 7.4) was conjugated by electrostatic interaction.
  • NaCl was added to the final micelle suspension at a concentration of 150 mM.
  • FIG. 15 shows the results of evaluating the release of sgRNA of a co-encapsulated micelle (co-encap. Micelle) co-encapsulated with Cy5-sgRNA and Cy3-Cas9 mRNA and a Cy5-sgRNA micelle (sgRNA micelle). .. Each micelle was diluted 1-100-fold with 10 mM phosphate buffer and incubated at 37 ° C. for 30 minutes. Then, the release of sgRNA was evaluated by agarose gel electrophoresis. As shown in FIG. 15, the co-emulsified micelles released less sgRNA at any dilution rate than the sgRNA micelles. This result shows that sgRNA is stably retained in the micelle even in the co-emulsified micelle using PEG-PLys.
  • PIC micelle 1 Polyion complex micelle (PIC micelle) 11 Cas protein mRNA (Cas mRNA) 12 Guide RNA P block copolymer b1 cationic polymer block b2 hydrophilic polymer block

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