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WO2022098141A1 - Protéine d'édition de gène dans laquelle un acide aminé non naturel est introduit de manière sélective quant aux résidus et procédé d'édition de gène l'utilisant - Google Patents

Protéine d'édition de gène dans laquelle un acide aminé non naturel est introduit de manière sélective quant aux résidus et procédé d'édition de gène l'utilisant Download PDF

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WO2022098141A1
WO2022098141A1 PCT/KR2021/015996 KR2021015996W WO2022098141A1 WO 2022098141 A1 WO2022098141 A1 WO 2022098141A1 KR 2021015996 W KR2021015996 W KR 2021015996W WO 2022098141 A1 WO2022098141 A1 WO 2022098141A1
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Prior art keywords
protein
gene editing
cas9
compound
gene
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Korean (ko)
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정현정
양승주
임산해
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Korea Advanced Institute of Science and Technology KAIST
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Korea Advanced Institute of Science and Technology KAIST
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • It relates to a gene editing protein into which a non-natural amino acid is residue-selectively introduced, and a gene editing method using the same.
  • Gene therapy products can precisely control disease target genes, and as a versatile technology, they have great advantages over existing protein or small molecule drugs. is being studied The gene therapy market is expected to grow at a CAGR of 33.3% from $584 billion (about $658.4 billion) in 2016 to $4.402 billion (about $5 trillion) in 2023. Gene therapy products for cancer and rare diseases account for more than 60% of the total market, followed by neurological diseases, cardiovascular diseases, and infectious diseases.
  • virus-based delivery methods such as retrovirus, adeno-associated virus (AAV), and lentiviruses show high delivery efficiency, making it difficult to develop targeting various diseases.
  • AAV adeno-associated virus
  • lentiviruses show high delivery efficiency, making it difficult to develop targeting various diseases.
  • Editas of the United States has invested heavily in in vivo gene therapy technology and developed a therapeutic agent in partnership with Allergan Pharmaceuticals targeting ophthalmic diseases.
  • it is based on a virus-based in vivo genome editing technology developed for patients with a rare congenital retinal disease called Leber congenital amaurosis type 10 (LCA).
  • LCA Leber congenital amaurosis type 10
  • This technology is applied by directly injecting adeno-associated virus (AAV) into the eye to correct the splicing error of the CEP290 gene.
  • AAV adeno-associated virus
  • the program was delayed due to difficulties in the AAV manufacturing process, but at the end of 2018, the FDA approved a phase 1/2 dose escalation trial. It is reported that AAV can be applied relatively safely compared to other viruses due to its small size, but due to the large size of the CRISPR expression vector, the packaging efficiency for AAV is very low, making development difficult.
  • the virus-based delivery method has limitations such as immune side effects and carcinogenicity in the human body, so a non-viral delivery method is preferred.
  • the non-viral delivery method has a great advantage in terms of safety when administered in vivo, but shows a low delivery efficiency into target cells and cells, so a carrier material to promote delivery must be used.
  • a carrier material to promote delivery must be used.
  • cationic polymers, lipids, inorganic nanoparticles, cell-penetrating peptides, and dendrimers have been actively studied as carrier materials, many problems remain to be solved due to cytotoxicity and low loading efficiency.
  • ribonucleic acid protein is composed of Cas9 protein and single guide RNA (sgRNA).
  • sgRNA single guide RNA
  • CRISPR/Cas9 technology is the largest, occupying 53.8% of the total with $1.95 billion in 2018, and is expected to grow by 14.7% by 2023 to reach $3.87 billion, accounting for 54.2% of the gene editing market. is expected to be
  • Ribonucleic acid protein is the most direct delivery method and has the highest potential for development as a therapeutic agent due to its transient effect, and a method for intracellular delivery with a previously developed carrier for gene delivery is being studied.
  • Intellia of the United States is focusing on a therapeutic technology based on the delivery of Cas9 and guide RNA ribonucleic acid using lipid nanoparticles (LNP), which have been studied for a long time for siRNA delivery.
  • Intellia is jointly developing a treatment with Regeneron for a rare liver disease called transthyretin amyloidosis using its proprietary LNP technology, and has published preclinical results in primates.
  • Toolgen is investing heavily in the development of therapeutic agents for hemophilia, peripheral nerve disease called Charcot-Marie-Tooth, and type 10 Levers congenital amaurosis disease through in vivo delivery by AAV.
  • Toolgen in collaboration with Seoul National University, reported that the editing effect by HDR was also shown in a mouse model of Levers congenital amaurosis disease by delivering CRISPR-expressing AAV.
  • CRISPR RNP was administered for the treatment of Charcomaritus, and the results of indel editing and therapeutic efficacy were announced in a mouse model in 2019.
  • the Cas9 protein has a large molecular weight and unstable structure, it is difficult to mount on a carrier, so intracellular delivery and calibration efficiency is low, and there is a problem of carrier toxicity.
  • a method of chemically modifying the Cas9 protein which is a component of the gene editing tool, can be used.
  • the carrier is delivered to a non-target site and the problem of toxicity can be minimized. It has a great advantage as a platform technology because it can be maintained high.
  • bio-orthogonal reactive groups can be imparted to the target protein, and various biomolecular materials such as carriers for delivery and ligands can be freely conjugated.
  • the reaction is possible in situ, so it has high utilization value for delivery into target cells.
  • the present invention is a technology capable of high-efficiency and highly selective gene editing by residue-selectively introducing a non-natural amino acid into Cas9, a gene editing protein, to give a bioorthogonal reactive group, conjugating a biomaterial in situ and delivering it into the cell.
  • One aspect provides a gene editing protein into which a non-natural amino acid has been residue-selectively introduced.
  • composition for gene editing comprising the gene editing protein and a compound having an alkyne or cycloalkyne group or a salt thereof.
  • Another aspect comprises the steps of residue-selectively introducing a non-natural amino acid into a gene editing protein
  • It provides a gene editing method comprising the step of treating or administering the gene editing protein and a compound having an alkyne or cycloalkyne group or a salt thereof to cells isolated from an individual.
  • One aspect relates to gene editing proteins into which non-natural amino acids have been residue-selectively introduced.
  • the non-natural amino acid may be an amino acid having an azide group, specifically, AHA (4-Azido-homoalanine) or a salt thereof.
  • AHA (4-Azido-homoalanine) or a salt thereof may be derived from nature or may be synthesized using a known organic synthesis method.
  • the AHA (4-Azido-homoalanine) or a salt thereof may be a non-protein compound, a peptide, an extract of a plant-derived tissue or cell, or a product obtained by culturing a microorganism (eg, bacteria or fungi, and particularly yeast). .
  • a microorganism eg, bacteria or fungi, and particularly yeast.
  • salt refers to a salt prepared using a specific compound according to an aspect and a relatively non-toxic acid or base.
  • the “salt” may be a “pharmaceutically acceptable salt”.
  • pharmaceutically acceptable is meant to exhibit non-toxic properties to cells or humans exposed to the compound.
  • pharmaceutically acceptable salt refers to a salt prepared using a specific compound according to one aspect and a relatively non-toxic acid or base.
  • the base addition salt can be obtained by contacting the neutral form of the compound with a sufficient amount of the base in pure solution or in a suitable inert solvent.
  • Pharmaceutically acceptable base addition salts include salts of sodium, potassium, calcium, ammonium, organic amines, or magnesium or similar salts.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent.
  • Pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, hydrogen carbonate ion, phosphoric acid, phosphate monohydrogen ion, phosphate dihydrogen ion, sulfuric acid, hydrogen sulfate ion, hydroiodic acid or phosphorous acid; and salts of organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid and salts of amino acids (eg, arginine) and salts of organic acids such as glucuronic acid are also included.
  • inorganic acids such as hydrochloric acid, hydrobromic acid,
  • the pharmaceutically acceptable salt may be synthesized from a parent compound containing an acidic or basic moiety by a conventional chemical method.
  • such salts are prepared by reacting the free acid or form of the base of these compounds with a stoichiometrically appropriate amount of the base or acid, in water or in an organic solvent or in a mixture of the two.
  • non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred.
  • the "residue-specifically introduced” may mean that a specific type of residue in the amino acid sequence of a target protein or polypeptide is selectively modified.
  • the modification may be, for example, at least one selected from the group consisting of substitution, deletion, and insertion.
  • protein refers to a polymer composed of two or more amino acids linked by amide bonds (or peptide bonds).
  • the gene editing protein may be a modified one of Met (Methionine) in the amino acid sequence of the wild-type protein, specifically, Met (Methionine) in the amino acid sequence of the wild-type protein to AHA (4-Azido-homoalanine) may be substituted.
  • the gene editing protein may be one used in a CRISPR/Cas9 system, specifically, a Cas protein, and more specifically, a Cas9 protein.
  • the gene editing protein has an amino acid sequence of the gene editing protein and each of about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 92% or more, about 95% or more, about It may comprise a protein having at least 97%, at least about 98%, or at least about 99% sequence homology.
  • homology is intended to indicate a degree of similarity with a wild-type amino acid sequence, and the comparison of such homology can be performed using a comparison program well known in the art, and the homology between two or more sequences is It can be calculated as a percentage (%).
  • a protecting group may be bonded to the C-terminus.
  • the protecting group may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group or polyethylene glycol (PEG), but the modification of the gene editing protein, particularly the stability of the gene editing protein As long as it is a component capable of enhancing , it may be included without limitation.
  • stability may refer to storage stability (eg, room temperature storage stability) as well as in vivo stability to protect the polypeptide of the present invention from attack by proteolytic enzymes in vivo.
  • the gene editing protein may additionally include an amino acid sequence prepared for a specific purpose for a targeting sequence, a tag, and a labeled residue.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • a tracr-mate sequence including “direct repeats” and tracrRNA-processing portion direct repeats in the context of an endogenous CRISPR system
  • a guide sequence also referred to as a "spacer” in the context of an endogenous CRISPR system
  • a guide RNA or CRISPR locus refers to transcripts and other elements involved in the expression of, or inducing activity of, CRISPR-associated (Cas) genes, including other sequences and transcripts from In some embodiments, one or more elements of the CRISPR system are from a type I, type II, or type III CRISPR system.
  • one or more elements of the CRISPR system are derived from a particular organism comprising the endogenous CRISPR system, eg, Streptococcus pyogenes .
  • CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of the target sequence (also referred to as protospacers in the context of endogenous CRISPR systems).
  • a "target sequence” or “target gene” refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence enhances the formation of the CRISPR complex.
  • the target sequence may comprise any polynucleotide, eg, a DNA or RNA polynucleotide.
  • the target sequence is located in the nucleus or cytoplasm of a cell.
  • the target sequence may be present in an organelle of a eukaryotic cell, such as a mitochondrion or chloroplast.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx3, Csx10, Csx3, Csx10, Csx1, Csf2, Csf3, Csf4, homologue
  • the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be obtained from the SwissProt database under accession number Q99ZW2.
  • the unmodified CRISPR enzyme eg, Cas9, has DNA cleavage activity.
  • the CRISPR enzyme or gene editing protein is a Cas9 protein, wherein the Cas9 protein is a Streptococcus pyogenes -derived Cas9 protein, a Campylobacter jejuni -derived Cas9 protein, a Streptococcus thermophilic It may be at least one Cas9 protein selected from the group consisting of a Cas9 protein derived from Streptococcus thermophiles , a Cas9 protein derived from Streptococcus aureus and a Cas9 protein derived from Neisseria meningitidis , and specifically To Streptococcus pyogenes ( Streptococcus pyogenes ) It may be a Cas9 protein, more specifically, a protein consisting of the amino acid sequence of SEQ ID NO: 1 may be a modified protein, for example, consisting of the amino acid sequence of SEQ ID NO: 1 The protein may be a protein consisting of the modified
  • the Cas9 protein may include a nuclear localization sequence or signal (NLS) at 5'- or 3'- or both terminal portions of the Cas9 protein for localization in the nucleus in a eukaryotic cell, wherein the NLS is It can be one or more.
  • NLS nuclear localization sequence or signal
  • nuclear localization sequence or signal refers to an amino acid sequence that serves to transport a specific substance (eg, protein) into a cell nucleus, and is generally a nuclear pore (Nuclear Pore). ) through the cell nucleus.
  • a specific substance eg, protein
  • nuclear pore Nuclear Pore
  • RNA-guided CRISPR clustered regularly interspaced short palindrome repeats
  • CRISPR clustered regularly interspaced short palindrome repeats
  • sgRNA single guide RNA
  • Cas9 (or Cpf1) protein refers to an essential protein element in the CRISPR/Cas9 system, and information on the Cas9 (or Cpf1) gene and protein can be obtained from GenBank of the National Center for Biotechnology Information (NCBI). However, the present invention is not limited thereto.
  • the CRISPR-associated gene encoding the Cas (or Cpf1) protein is known to exist in about 40 or more different Cas (or Cpf1) protein families, and according to the specific combination of the cas gene and the repeat structure, 8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, and Mtube) can be defined. Therefore, each of the CRISPR subtypes can form a repeating unit to form a polyribonucleotide-protein complex.
  • the DNA may generally (but not necessarily) include regulatory elements (eg, a promoter) operable in a target cell.
  • the promoter for Cas9 expression may be, for example, a CMV, EF-1 a, EFS, MSCV, PGK, or CAG promoter.
  • a promoter for gRNA expression may be, for example, a HI, EF-la, tRNA or U6 promoter.
  • the sequence of the gene encoding Cas9 may include a nuclear localization signal (NLS) (e.g., SV40 NLS).
  • the promoter may have tissue specificity or cell specificity.
  • chimeric RNA refers to a guide sequence, a tracr sequence and / or a polynucleotide sequence comprising a tracr mate sequence.
  • guide sequence refers to a sequence of about 20 bp within a guide RNA that directs a target site, and may be used interchangeably with the terms “guide” or “spacer”.
  • tracr mate sequence may be used interchangeably with the term “direct repeat(s)”.
  • the guide RNA may be composed of two RNAs, that is, CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA), or a single-stranded RNA comprising portions of crRNA and tracrRNA and hybridizing with the target DNA. (single-chain RNA, sgRNA).
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and induce sequence-specific binding of the CRISPR complex to the target sequence.
  • a portion of the guide RNA nucleotide sequence may be modified in order to modify/enhance the function of the guide RNA.
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using an appropriate alignment algorithm, is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more.
  • Optimal alignment can be determined using any algorithm suitable for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burroughs- Algorithms based on the Burrows-Wheeler Transform (eg Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies) , ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn) and Maq (available at maq.sourceforge.net).
  • any algorithm suitable for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, the Burroughs- Algorithms based on the Burrows-Wheeler Transform (eg Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies) , ELAND (Illumina, San Diego, CA), SOAP (available at
  • guide sequences is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40 , 45, 50, 75 or more nucleotides in length. In some embodiments, the guide sequence is about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length.
  • the ability of the guide sequence to induce the sequence-specific binding of the CRISPR complex of can be assessed by any suitable assay, for example, a component of the CRISPR system sufficient to form a CRISPR complex comprising the guide sequence being tested.
  • cleavage of target polynucleotide sequence is CRIS containing target sequence, test guide sequence and control guide sequence different from test guide sequence. It can be assessed in vitro by providing the components of the PR complex and comparing the rates of binding or cleavage between test and control guide sequence reactions at the target sequence.
  • Other assays are possible and will be readily available to those skilled in the art.
  • the guide sequence can be selected to target any target sequence.
  • the target sequence is a sequence within the genome of a cell.
  • Exemplary target sequences include those that are unique in the target genome.
  • a unique target sequence in the genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNXGG, where NNNNNNNNNNXGG (N is A, G, T or C; X is any ) has a single presence in the genome.
  • a unique target sequence in a genome may comprise a Streptococcus pyogenes Cas9 target site of the form MMMMMMMNNNNNNNNNNNNNXGG, wherein NNNNNNNNNXGG (N is A, G, T or C; X may be any) is a single in the genome has the existence of
  • the unique target sequence in the genome may comprise a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNNXXAGAAW, wherein NNNNNNNNNNXXAGAAW (N is A, G, T or C; X can be any; ; W is A or T) has a single occurrence in the genome.
  • a unique target sequence within a genome may comprise a Streptococcus thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNNNXXAGAAW, wherein NNNNNNNNNXXAGAAW (N is A, G, T or C; X can be anything; W is A or T) has a single occurrence in the genome.
  • a unique target sequence in the genome may comprise a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNNNXGGXG, wherein NNNNNNNNNNNNXGGXG (N is A, G, T or C; X may be anything) has a single presence in the genome.
  • a unique target sequence in a genome may comprise a Streptococcus pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNNNXGGXG, wherein NNNNNNNNNXGGXG (N is A, G, T, or C; X may be any) is single in the genome has the existence of In each of these sequences, “M” can be A, G, T or C.
  • the gene editing protein may be modified through conjugation with a compound having an alkyne or cycloalkyne group or a salt thereof.
  • the compound or salt thereof having an alkyne or cycloalkyne group may be one capable of a click chemical reaction with an azide group in the gene editing protein, and specifically, a compound having a DBCO (Dibenzylcyclooctyne) group or a salt thereof may be.
  • DBCO Dibenzylcyclooctyne
  • the compound having an alkane or cycloalkyne group may be a compound represented by the following formula (2):
  • salt may be a “pharmaceutically acceptable salt”, and specifically may be within the aforementioned range.
  • the compound or salt thereof having an alkyne or cycloalkyne group may be combined with an aptamer, an antibody, or a fragment thereof.
  • the aptamer, antibody, or fragment thereof is bound for a specific target (including antigen), and through this, the gene editing protein can be used for precise editing of a specific gene in a specific cell.
  • the compound or salt thereof having an alkyyl or cycloalkyne group may be bound to an aptamer, an antibody, or a fragment thereof that targets a specific cell, and the aptamer, the antibody, or a fragment thereof targets a specific Through endocytosis in a cell, a gene editing protein conjugated to the compound having an alkine or cycloalkyne group or a salt thereof is incorporated into the cell to edit a specific gene.
  • compositions for gene editing comprising the gene editing protein and a compound having an alkyne or cycloalkyne group or a salt thereof.
  • the "compound having an alkyne or cycloalkyne group”, “salt”, “gene editing”, etc. may be within the above-mentioned range, for example, the compound having an alkyne or cycloalkyne group or a salt thereof is the gene It may be possible to react with an azide group in the editing protein and click chemical reaction.
  • the compound having an alkyne or cycloalkyne group may be, for example, a compound having a DBCO (Dibenzylcyclooctyne) group, and may be a compound represented by the following Chemical Formula 2:
  • the "compound having an alkane or cycloalkyne group” may be combined with an aptamer, an antibody, or a fragment thereof.
  • the gene editing protein and the compound having an alkane or cycloalkyne group or a salt thereof may be conjugated, and the gene editing protein and the alkane or cycloalkyne group in the composition may be conjugated.
  • the compound or salt thereof may be pre-conjugated.
  • the compound or salt thereof having an alkyyl or cycloalkyne group may be bound to an aptamer, an antibody, or a fragment thereof that targets a specific cell, and the aptamer, the antibody, or a fragment thereof is contained in a specific target cell Through endocytosis, a gene editing protein conjugated to the compound having an alkyne or cycloalkyne group or a salt thereof is incorporated into a cell to edit a specific gene.
  • Another aspect comprises the steps of residue-selectively introducing a non-natural amino acid into a gene editing protein
  • It relates to a gene editing method comprising the step of treating or administering the gene editing protein and a compound having an alkyne or cycloalkyne group or a salt thereof to cells isolated from the subject.
  • the “gene editing protein”, "unnatural amino acid”, “residue-selectively introduced”, “a compound having an alkyne or cycloalkyne group or a salt thereof” and the like may be within the above-described range.
  • the non-natural amino acid may be AHA (4-Azido-homoalanine)
  • the gene editing protein may be a Cas9 protein
  • the compound having an alkyne or cycloalkyne group is DBCO (Dibenzylcyclooctyne) It may be a compound having a group, and the compound or salt thereof having an alkyne or cycloalkyne group may be bound to an aptamer, an antibody, or a fragment thereof.
  • DBCO Dibenzylcyclooctyne
  • the gene editing method comprises the steps of conjugating a compound having an alkine or cycloalkyne group or a salt thereof with the gene editing protein prior to treatment or administration to cells isolated from an individual. may include more.
  • the step of residue-selectively introducing the non-natural amino acid may be a step of substituting AHA (4-Azido-homoalanine) for Met (Methionine) in the amino acid sequence of the wild-type protein.
  • administration means introducing a predetermined substance to an individual by an appropriate method
  • individual means any living organism, including humans, mice, mice, livestock, etc., carrying the gene to be edited. As a specific example, it may be a mammal including a human.
  • the "cells isolated from an individual” may be cells isolated from an individual as well as cells subcultured from a cell isolated from an individual by a known culturing method, etc., and may be genetically, chemically or It may be a cell that has been physically manipulated or modified, or it may be a newly artificially synthesized or fabricated cell.
  • the route of administration is, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or work.
  • the gene editing protein and the compound having an alkyne or cycloalkyne group or a salt thereof are administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level depends on the type, severity, activity of the drug, and the drug in the patient. It can be determined according to factors including sensitivity, administration time, administration route and excretion rate, duration of treatment, concurrent drugs, and other factors well known in the medical field.
  • the gene editing protein and the compound having an alkyne or cycloalkyne group or a salt thereof may be administered at 0.001 to 1000 mg/kg/day, more specifically, at 0.1 to 100 mg/kg/day.
  • the administration may be administered once a day or may be administered in several divided doses.
  • the effective amount of the gene editing protein and the compound having an alkyne or cycloalkyne group or a salt thereof depends on the patient's age, sex , state, body weight, absorption of the active ingredient into the body, inactivation rate, excretion rate, disease type, and drugs used in combination, and may vary depending on the route of administration, severity of obesity, sex, weight, age, etc.
  • the gene editing protein and the compound having an alkyne or cycloalkyne group or a salt thereof may include an active ingredient alone, or may be provided by including one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the carrier may be, for example, colloidal suspension, powder, saline, lipid, liposome, microspheres or nano-spherical particles. They may form complexes with or be associated with a vehicle and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.
  • Lubricants sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, extenders, binders, wetting agents, and disintegrants commonly used when the gene editing protein and the compound having an alkyne or cycloalkyne group or salts thereof are formulated. It can be formulated using diluents or excipients such as release agents, surfactants, and the like.
  • Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the composition, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. can be mixed and prepared.
  • Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are commonly used simple diluents, may be included.
  • Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • injectable esters such as ethyl oleate.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin, glycero geratin, etc. may be used, and a known diluent or excipient may be used when preparing in the form of eye drops. there is.
  • the compound or salt thereof having an alkyyl or cycloalkyne group may be bound to an aptamer, an antibody, or a fragment thereof that targets a specific cell, and the aptamer, the antibody, or a fragment thereof is contained in a specific target cell Through endocytosis, a gene editing protein conjugated to the compound having an alkyne or cycloalkyne group or a salt thereof is incorporated into a cell to edit a specific gene.
  • One aspect provides a technology for residue-selectively introducing a non-natural amino acid into a gene editing protein to conjugate and form a complex to a biomaterial through a selective click chemistry reaction and deliver it into a cell.
  • it is possible to control the number of bioorthogonal reactors introduced into the gene editing protein, and in the process of delivering the gene editing protein into the cell, it causes a selective chemical reaction with other physiologically active substances in situ, resulting in high efficiency, high selective delivery and As it can cause CRISPR gene correction, it can be usefully utilized as a therapeutic agent for in vivo and ex vivo cell correction.
  • FIG. 1 shows a schematic diagram for the production and delivery of a Cas9 complex into which non-natural amino acids are residue-selectively introduced.
  • Figure 2 is the result of analyzing the bioorthogonal chemical reactivity in the lysate after the production of Cas9-AHA in B834.
  • Figure 6 shows the results of the intracellular uptake test in SKBR3 (HER2 positive) and MCF7 (HER2 negative) (Ab: anti-HER2 monoclonal antibody, Blue: nuclear, Red: Cas9 / Cas9 (AHA)).
  • a non-natural amino acid Azdiohomoalanine (AHA) was residue-selectively introduced into Cas9 to produce a gene editing protein capable of a bioorthogonal reaction, and a biomaterial (eg, It was confirmed that intracellular delivery can be promoted by conjugating or co-treating a delivery promoting substance, a targeting ligand, a nucleic acid, etc., or co-delivery with a ligand substance.
  • AHA Azdiohomoalanine
  • AHA (4-Azido-homoalanine) has a structure similar to Methionine (Met) among the 20 essential amino acids, and has a structure in which an azide group is introduced instead of a methylsulfide group in the Met side chain, and is a compound represented by the following Chemical Formula 1:
  • AHA is residue-selectively introduced instead of Met into the amino acid sequence of proteins synthesized by tRNAMet and amino acyl tRNA synthetase.
  • the AHA introduced into the Cas9 protein has an azide functional group, so that various biomaterials can be conjugated by a chemoselective reaction.
  • the azide group is a functional group capable of ‘click chemical reaction’ and causes a very rapid and irreversible reaction with an alkane or cycloalkyne. Since this reaction occurs specifically in a complex in vivo environment, it is used to react with biomolecules in situ.
  • DBCO Dibenzylcyclooctyne
  • B834-(DE3) was cultured by adding Met or AHA, and the lysate obtained by dissolving it was reacted with DBCO-PEG4-BODIPY-FL at room temperature for 1 hour. was the same, and as a result of analyzing it through fluorescence imaging, it was the same as that of FIG. 2B. Through this, only the lysate obtained by adding AHA showed fluorescence, indicating that B834-(DE3) can produce protein by introducing AHA, and that the introduced proteins can be conjugated by click chemical reaction. .
  • cleavage activity was analyzed using a synthetic DNA template and sgRNAs.
  • PLK1 poly-like kinase 1
  • sgRNA-1 SEQ ID NO: 3
  • sgRNA-2 SEQ ID NO: 4
  • DNA template targeted by sgRNA-1 SEQ ID NO: 5
  • DNA template targeted by sgRNA-2 SEQ ID NO: 6
  • the Cas9 protein complexed with sgRNA-1 cut the target DNA with an efficiency of 97.8%, and the Cas9(AHA) protein cut with an efficiency of 95.5%, showing similar activity.
  • the Cas9 protein cleaved the target DNA with an efficiency of 86.7%, and the Cas9 (AHA) protein cleaved with an efficiency of 87.0%. This indicates that the introduction of AHA does not degrade the enzymatic function of the Cas9 protein.
  • the synthesized Ab-DBCO was treated with SKBR3 (HER2 positive) and HEK293T (HER2 negative) and incubated for 10 minutes, followed by treatment with Cas9 (AHA) and Cas9 as a control, and further incubated for 4 hours. After incubation, cell fixation and actin and nuclei were stained for imaging analysis, and then analyzed by confocal microscopy.
  • Cas9 was located in the plasma membrane and cytosol only when SKBR3 was treated with Ab-DBCO and Cas9 (AHA). It can be seen that Cas9 (AHA) and Ab-DBCO cause a bioorthogonal reaction in situ during cell processing, and target-specific cell delivery is promoted by endocytosis ( FIG. 5 ). That is, it was confirmed that target-specific delivery into cancer cells was possible by co-delivery of the ligand material modified with Cas9-AHA and DBCO.
  • the antibody-conjugated Cas9 (AHA) could be internalized in the target cell.
  • the DBCO-modified antibody (HER2-SS- DBCO) was synthesized.
  • Cas9 protein and Cas9 (AHA) protein were reacted with AF647-NHS ester in a 1:1 molar ratio to facilitate imaging analysis.
  • -SS- is a disulfide bond that can be cleaved by a reducing agent such as glutathione.
  • HER2 Ab-SS-Cas9(AHA) internalized in target cells
  • Cas9(AHA) is separated, and eventually free Cas9(AHA) ) is transmitted to the nucleus.
  • the synthesized HER2 Ab-SS-DBCO was treated with SKBR3 (HER2 positive) and MCF7 (HER2 negative) and incubated for 10 minutes, followed by treatment with Cas9 (AHA) and Cas9 as a control, and further incubated for 4 hours.
  • HER2-SS-DBCO and Cas9 (AHA) and Cas9 were reacted for 1 hour at room temperature, and then treated with the cells 4 incubated for hours. After incubation, cell fixation and actin and nuclei were stained for imaging analysis, and then analyzed by confocal microscopy.
  • HER2 Ab-SS-DBCO Ab-SS-DBCO
  • SKBR3 HER2 positive
  • MCF7 HER2 negative
  • PLK1 gene was Complex with sgRNA (sgPLK1: SEQ ID NO: 7; 5'-GAAATTAATACGACTCACTATAGG TACCTACGGCAAATTGTGCT GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3'; the above bolded spacer sequence) was incubated with Cas9 complexed for 48 hours.
  • sgPLK1 SEQ ID NO: 7; 5'-GAAATTAATACGACTCACTATAGG TACCTACGGCAAATTGTGCT GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT-3'; the above bolded spacer sequence
  • Cas9(AHA) complex and Ab-SS-DBCO were pre-conjugated by a click chemistry reaction (Cas9(AHA)-SS-Ab), and the target cells were treated and cultured for 48 hours.
  • Cas9(AHA)-SS-Ab a click chemistry reaction
  • Cas9(AHA) and DBCO-introduced antibodies can be successfully conjugated in one step both in situ and in vitro , and are delivered to target cells by antibody-mediated endocytosis, which eventually induces gene editing. confirmed that
  • the Cas9-AHA protein of the present invention can be purified with high yield and purity by a simple method, and can bind biomolecules quickly and specifically in an in vivo environment, and the Cas9-AHA protein is a target It was confirmed that it can effectively cut DNA.
  • This Cas9-AHA can easily bind various biomolecules, and efficient and effective Cas9 protein complex synthesis, intracellular delivery, and gene editing of target DNA can be expected using this Cas9-AHA.

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Abstract

Un aspect se rapporte à une protéine d'édition de gène dans laquelle un acide aminé non naturel est introduit de manière sélective quant aux résidus, et à un procédé d'édition de gène l'utilisant. En particulier, selon un aspect, le nombre de réacteurs bioorthogonaux introduits dans la protéine d'édition de gène peut être régulé, et des réactions chimiques sélectives, in situ, avec d'autres substances physiologiquement actives sont provoquées lors du processus d'administration d'une protéine d'édition de gène dans une cellule, de telle sorte qu'une administration hautement efficace, hautement sélective et une correction de gène CRISPR peuvent se produire, permettant ainsi à la protéine d'édition de gène d'être utilisée utilement en tant qu'agent thérapeutique de correction cellulaire in vivo et ex vivo.
PCT/KR2021/015996 2020-11-06 2021-11-05 Protéine d'édition de gène dans laquelle un acide aminé non naturel est introduit de manière sélective quant aux résidus et procédé d'édition de gène l'utilisant Ceased WO2022098141A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2017205336A1 (fr) * 2016-05-23 2017-11-30 The Trustees Of Columbia University In The City Of New York Dérivés nucléotidiques et leurs méthodes d'utilisation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017205336A1 (fr) * 2016-05-23 2017-11-30 The Trustees Of Columbia University In The City Of New York Dérivés nucléotidiques et leurs méthodes d'utilisation

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EUNHA KIM, HEEBEOM KOO: "Biomedical applications of copper-free click chemistry: in vitro , in vivo , and ex vivo", CHEMICAL SCIENCE, ROYAL SOCIETY OF CHEMISTRY, UNITED KINGDOM, vol. 10, no. 34, 28 August 2019 (2019-08-28), United Kingdom , pages 7835 - 7851, XP055740836, ISSN: 2041-6520, DOI: 10.1039/C9SC03368H *
KIM JOO-GANG, JUNG HYUN-JUNG: "Bioorthogonal functionalization of Cas9 protein with bioactive molecule via click chemistry", APPLIED CHEMISTRY FOR ENGINEERING, HAN'GUK KONGOP HWAHAKHOE, KOREAN SOCIETY OF INDUSTRIAL AND ENGINEERING CHEMISTRY, KR, vol. 22, no. 1, 1 January 2018 (2018-01-01) - 4 May 2018 (2018-05-04), KR , XP009536562, ISSN: 1225-0112 *
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