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WO2021085580A1 - Polynucléotide et son utilisation - Google Patents

Polynucléotide et son utilisation Download PDF

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Publication number
WO2021085580A1
WO2021085580A1 PCT/JP2020/040738 JP2020040738W WO2021085580A1 WO 2021085580 A1 WO2021085580 A1 WO 2021085580A1 JP 2020040738 W JP2020040738 W JP 2020040738W WO 2021085580 A1 WO2021085580 A1 WO 2021085580A1
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polynucleotide
sequence
cre
encoding
recombinant
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Japanese (ja)
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雄二 恒川
文雄 松崎
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RIKEN
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)

Definitions

  • the present invention relates to polynucleotides, vectors, gene recombination kits, Cre modified proteins or fusion proteins thereof, and gene recombination methods.
  • recombinase a gene recombination technology using a recombinant enzyme
  • An example of such a gene recombination technique is a Cre / loxP system using Cre recombinase and loxP sequences.
  • the recombinant enzyme and a promoter sequence that functions specifically at a site are used in combination to form a site-specific group rather than the whole body. Recombination can occur (see, for example, Non-Patent Documents 1 and 2).
  • the present inventors have assembled even in cells in which the recombinant enzyme should not be expressed when a donor DNA containing a polynucleotide encoding a recombinant enzyme is introduced into the cell.
  • the present inventors translated the transcript after the polynucleotide encoding the recombinant enzyme contained in the donor DNA before being knocked in was transcribed non-specifically to the cell type. I faced a new challenge of In the present specification, this phenomenon is referred to as "leak".
  • This phenomenon of "leakage” can be a particular problem when a large amount of donor DNA is used to introduce the recombinant enzyme into cells.
  • the de novo knock-in method developed by the present inventors (Tsunekawa Y et al. (2016) "Developing a de novo targeted knock-in method based on in utero electroporation into the mammal brain," Development, Vol.143 (No) .17), pp.3216-3222.)
  • the HITI method (Suzuki K, Tsunekawa Y, et al. (2016) "In vivo genome editing via CRISPR / Cas9 mediated mammal-independent targeted integration," Nature, Vol.540 (Issue 7331), pp.144-149.) Can be a particular problem.
  • One aspect of the present invention is to provide a polynucleotide capable of reducing the occurrence of leaks.
  • the present inventors have found that the occurrence of leaks can be reduced by controlling the step of translation among the two steps of transcription and translation involved in the leak, and complete the present invention. I arrived. More specifically, we found that the occurrence of leaks can be reduced by substituting at least some of the translation initiation codons present in the polynucleotide encoding the recombinant enzyme with untranslation initiation codons. We have found and completed the present invention.
  • the present invention includes the following configurations. ⁇ 1> The polynucleotide shown in any of the following (G1) to (G4): (G1) (i) In a polynucleotide encoding a recombinant enzyme, a protein consisting of a polynucleotide in which at least one translation initiation codon has been replaced with a non-translation initiation codon, and (ii) a protein having recombinant activity.
  • the polynucleotide encoding (G2) (i) At least one translation initiation in a polynucleotide consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added to a polynucleotide encoding a recombinant enzyme.
  • a polynucleotide consisting of a polynucleotide whose codon has been replaced with an untranslated start codon and (ii) encoding a protein having recombinant activity; (G3) (i) Consists of a polynucleotide in which at least one start codon is replaced with a non-translation start codon in a polynucleotide having 90% or more sequence identity with the polynucleotide encoding the recombinant enzyme.
  • a polynucleotide encoding a protein having recombinant activity (G4) (i) In a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a DNA sequence complementary to the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is untranslated.
  • a polynucleotide consisting of a polynucleotide substituted with a codon and (ii) encoding a protein having recombinant activity (ii) The polynucleotide according to ⁇ 1>, wherein the translation start codon is ATG, CTG, TTG, GTG, or ATA.
  • ⁇ 3> The polynucleotide according to ⁇ 1> or ⁇ 2>, wherein the recombinant enzyme is Cre, FLP, Dre, Tre, or a variant thereof.
  • ⁇ 4> A vector containing the polynucleotide according to any one of ⁇ 1> to ⁇ 3>.
  • ⁇ 5> The vector according to ⁇ 4>, further comprising a splicing acceptor sequence on the 5'end side of the polynucleotide.
  • ⁇ 6> A genetic recombination kit comprising the polynucleotide according to any one of ⁇ 1> to ⁇ 3> or the vector according to ⁇ 4> or ⁇ 5>.
  • a genetic recombination method comprising the step of introducing the polynucleotide according to any one of ⁇ 1> to ⁇ 3> or the vector according to ⁇ 4> or ⁇ 5> into a cell or a non-human subject.
  • a polynucleotide capable of reducing the occurrence of leaks is provided.
  • FIG. 1 It is a schematic diagram comparing the conventional donor DNA and the donor DNA in one embodiment of the present invention. It is a schematic diagram explaining the vector which concerns on one Embodiment of this invention and the method of using it. It is a schematic diagram which shows the composition of the donor DNA used in Example 1.
  • FIG. It is a figure which shows the result of Example 1.
  • FIG. It is a schematic diagram showing Cre1 to Cre6 (wild type Cre and a partial peptide thereof) produced in Example 2. It is a figure which shows the result of Example 2.
  • FIG. It is a figure which shows the result of Example 3.
  • FIG. It is a schematic diagram which shows the composition of the donor DNA used in Example 4 and knockin sight of the donor DNA. It is a figure which shows the result of Example 4.
  • Example 7 It is a schematic diagram which shows the composition of the donor DNA used in Example 5 and knockin sight of the donor DNA. It is a figure which shows the result of Example 5. It is a schematic diagram which shows the composition of the donor DNA used in Example 6 and knockin sight of the donor DNA. It is a figure which shows the result of Example 6. It is the result of Example 7, and is the figure which shows the EGFP expression rate of a negative control. It is the result of Example 7, and is the figure which shows the EGFP expression rate of a positive control. It is the result of Example 7, and is the figure which shows the EGFP expression rate in the group which used the wild type Cre. It is the result of Example 7, and is the figure which shows the EGFP expression rate in the group which used Opti Cre2. It is a figure which shows the result of Example 7. It is a figure which shows the result of Example 8.
  • polynucleotide is DNA
  • T thymine
  • U uracil
  • the upper panel of FIG. 1 represents the donor DNA 10 in the prior art.
  • the donor DNA 10 contains a polynucleotide 1 encoding a recombinant enzyme.
  • the polynucleotide 1 encoding the recombinant enzyme contains a translation initiation codon 5 (such as ATG).
  • the recombinant enzyme is released from the donor DNA 10 (polynucleotide 1 encoding the recombinant enzyme) even when the donor DNA 10 is not knocked in. It will be expressed. This phenomenon is referred to herein as a "leak".
  • the lower panel of FIG. 1 represents the donor DNA 10a in one embodiment of the present invention.
  • the donor DNA 10a contains the modified polynucleotide 1a. Specifically, in the modified polynucleotide 1a, at least one translation initiation codon 5 is replaced with the untranslation initiation codon 5a in the polynucleotide 1 encoding the recombinant enzyme. According to this configuration, leaks can be reduced at the translation level rather than at the transcription level (see Examples 1 and 3 for the effectiveness of the leak reduction strategy at the translation level).
  • reducing leakage means, for example, that the donor DNA 10a in one embodiment of the present invention is a recombinant enzyme derived from a donor DNA that has not been knocked in, rather than the donor DNA 10 of the prior art. It represents a state in which the expression level is reduced.
  • the expression level of the recombinant enzyme derived from the donor DNA 10a in the non-knocked state is 50% or less of the expression level of the recombinant enzyme derived from the donor DNA 10 of the prior art in the non-knocked state. 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 3% or less, 1% or less, or 0%.
  • the expression level of the recombinant enzyme derived from the donor DNA 10a in the non-knocked state and the expression level of the recombinant enzyme derived from the donor DNA 10 in the non-knocked state were measured by, for example, the method used in the examples. obtain. Specifically, it can be measured by introducing donor DNA10 or donor DNA10a into the cell without Cas9 (or its expression vector) and measuring the expression level of the recombinant enzyme. Methods for quantifying and comparing the expression levels of recombinant enzymes are well known in the art.
  • the modified polynucleotide 1a encodes a protein having recombinant activity. That is, the translated product of the modified polynucleotide 1a has recombinant activity.
  • the recombinant activity of the translated product of the modified polynucleotide 1a is 50% or more, 60% or more, 70% of the recombinant activity of the translation product of the polynucleotide 1 encoding the recombinant enzyme. Above, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
  • the recombination activity of the translation product of the modified polynucleotide 1a may be 100% or more of the recombination activity of the translation product of the polynucleotide 1 encoding the recombinant enzyme.
  • Whether or not the translated product of the modified polynucleotide 1a has recombinant activity can be confirmed by an assay common in the art (see Example 2 for a specific example). .. Alternatively, it is possible to estimate whether or not the translated product of the modified polynucleotide 1a has recombination activity by three-dimensional structural analysis. Further, a method for quantifying and comparing recombinant activity is a technique well known in the art (see Examples 7 and 8 for specific examples).
  • polynucleotide in the present specification includes RNA and DNA.
  • examples of polynucleotides in the form of RNA include mRNA.
  • examples of polynucleotides in the form of DNA include various DNA fragments, cDNAs, genomic DNAs.
  • RNA and DNA can have any structure (such as double-stranded or single-stranded).
  • the polynucleotide according to one embodiment of the present invention is the polynucleotide shown in any of the following (G1) to (G4).
  • G1 (I) In a polynucleotide encoding a recombinant enzyme, a polynucleotide consisting of a polynucleotide in which at least one translation initiation codon has been replaced with a non-translation initiation codon, and (ii) encoding a protein having recombinant activity. Polynucleotide that is.
  • G2 In a polynucleotide consisting of a nucleotide sequence in which one or several bases are substituted, deleted, inserted and / or added to a polynucleotide encoding a recombinant enzyme, at least one translation start codon is not present.
  • G3 In a polynucleotide having 90% or more sequence identity with the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is substituted with a non-translation initiation codon, and (i) ii) A polynucleotide encoding a protein having recombinant activity.
  • G4 In a polynucleotide that hybridizes under stringent conditions with a polynucleotide having a DNA sequence complementary to the polynucleotide encoding the recombinant enzyme, at least one translation initiation codon is replaced with a non-translation initiation codon.
  • Ii A polynucleotide encoding a protein having recombinant activity.
  • (G1) is intended as "a polynucleotide in which one or more translation initiation codons contained in the polynucleotide are replaced with non-translation initiation codons from a polynucleotide encoding a wild-type recombinant enzyme".
  • (G2) to (G4) refer to "one or more start codons contained in the polynucleotide from a variant or mutant polynucleotide of the polynucleotide encoding the wild-type recombinant enzyme”. It is intended as a "polynucleotide substituted with an untranslated start codon".
  • the polynucleotide encodes a protein having recombinant activity is determined by inserting the polynucleotide into a desired expression vector and then using the expression vector as a desired host. It can be confirmed by introducing and detecting whether or not gene recombination (for example, homologous recombination, non-homologous recombination) occurs in the host. If the gene has been recombined in the host, the polynucleotide can be determined to be "a polynucleotide encoding a protein having recombination activity". The detection of gene recombination may be carried out according to a known method (see, for example, Examples).
  • "several bases” are, for example, 50, 45, 40, 35, 30, 25, 20, 19, 19, 18, 17, 16, 15 There are 14, 14, 12, 11, 10, 10, 9, 8, 7, 6, 5, 4, 3, and 2 bases.
  • sequence identity is intended to be the proportion of the same number of bases.
  • sequence identity of the base sequence can be determined using, for example, BLASTN (Altschul SF (1990) "Basic local alignment search tool", Journal of Molecular Biology, Vol.215 (Issue 3), pp.403). -410).
  • Additions or deletions may be allowed to optimally align the base sequences to be compared.
  • the above-mentioned sequence identity is preferably 91% or more, more preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 95% or more, more preferably 96% or more, more preferably. Is 97% or more, more preferably 98% or more, and most preferably 99% or more.
  • hybridizing under stringent conditions means that hybridization is performed at 50 to 60 ° C. for 16 hours in a hybridization solution having a salt concentration of 6 ⁇ SSC to obtain a salt of 0.1 ⁇ SSC.
  • the condition for hybridization after washing in a solution of a concentration is sodium chloride: 150 mM and sodium citrate: 15 mM.
  • the translation initiation codon substituted with the untranslation initiation codon is not particularly limited.
  • Examples of translation start codons include ATG, CTG, TTG, GTG, ATA and the like. Of these, ATG is a typical translation initiation codon that is common to a wide range of species. On the other hand, CTG, TTG, GTG and ATA are atypical translation initiation codons that have been reported to function as translation initiation codons. Therefore, in the polynucleotide according to one embodiment of the present invention, it is preferable that one or more of the ATGs are replaced with non-translation initiation codons.
  • one or more of the ATGs are replaced with untranslated initiation codons, and one or more of the atypical translation initiation codons are untranslated initiation codons. It is more preferable that it is replaced with a codon.
  • the non-translation start codon in which the translation start codon is replaced is not particularly limited.
  • the methionine encoded by ATG is not encoded by codons other than ATG. Therefore, when ATG is replaced with an untranslated start codon, the ATG encoding methionine is inevitably replaced with a codon encoding an amino acid other than methionine.
  • the ATG is preferably replaced with a codon encoding leucine (TTA, TTG, CTT, CTC, CTA or CTG). This is because methionine and leucine have similar properties as amino acids (hydrophilic / hydrophobic, isoelectric point, three-dimensional structure, etc.). Which codon encoding leucine is replaced with ATG can be appropriately determined depending on, for example, the codon usage frequency of the organism into which the polynucleotide is introduced.
  • CTG, TTG, GTG and ATA can be replaced with non-translation start codons without changing the encoded amino acids.
  • CTG is replaced with CTC.
  • TTG is replaced with TTA.
  • GTG is replaced by GTA.
  • ATA is replaced with ATT.
  • the position of the translation initiation codon to be replaced with the non-translation initiation codon is not particularly limited as long as the desired translation product has recombination activity.
  • the position of the translation initiation codon substituted with the non-translation initiation codon is, for example, the amino terminal of the recombinant enzyme, the region on the amino terminal side of the recombinant enzyme, and the like.
  • the position may correspond to the region on the amino-terminal side of the central amino acid of the recombinant enzyme and the region on the amino-terminal side of the catalytic domain of the recombinant enzyme.
  • the number of translation initiation codons to be replaced with non-translation initiation codons is not particularly limited as long as the desired translation product has recombinant activity. For example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, or 50 or more translation initiation codons can be replaced with untranslated initiation codons. Leaks can be further reduced as the number of translation initiation codons replaced by untranslation initiation codons increases.
  • the upper limit of the number of translation initiation codons to be replaced with the untranslation initiation codon is not particularly limited, and may be 10, 20, 30, 40, 50, or 100.
  • the ratio of translation initiation codons replaced with non-translation initiation codons is not particularly limited as long as the translation product has recombinant activity. For example, 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 100% of translation initiation codons can be replaced with untranslated initiation codons. Leaks can be further reduced as the proportion of translation initiation codons replaced by untranslation initiation codons increases.
  • the upper limit of the ratio of translation initiation codons substituted with untranslated initiation codons is not particularly limited, and is 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less. , 80% or less, 90% or less, or 100% or less.
  • the recombinant enzyme is Cre, FLP, Dre, Tre, or a variant thereof (eg, a codon variant obtained by codon-optimizing a wild-type nucleotide sequence).
  • the recombinant enzyme is Cre, FLP, or Dre. More preferably, the recombinant enzyme is Cre.
  • recombinant enzymes are known recombinant enzymes, and the base sequences encoding them are also known.
  • the base sequence encoding the above recombinant enzyme is as follows. Cre: (SEQ ID NO: 1) FLP: (SEQ ID NO: 2) Dre: (SEQ ID NO: 3) Tre: (SEQ ID NO: 4).
  • Examples of the above-mentioned modified enzyme of the recombinant enzyme include VCre, SCre, FLPO, FLPE, DreE and the like. These variants are known variants, and the base sequences encoding them are also known. Specific nucleotide sequences are as follows: SEQ ID NO: 25 (VCre), SEQ ID NO: 26 (SCre), SEQ ID NO: 27 (FLPO), SEQ ID NO: 28 (FLPE).
  • the polynucleotide according to one embodiment of the present invention may be one or more selected from the group consisting of the following (G1') to (G4').
  • G1' (I) In the polynucleotide consisting of the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 4, at least one translation initiation codon is substituted with a non-translation initiation codon, and (ii) set.
  • the vector according to one aspect of the present invention contains the above-mentioned polynucleotide. By introducing such a vector into cells, leakage derived from the vector (for example, donor DNA) can be reduced.
  • vectors include phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors, virus-derived vectors (bacterial plasmids, bacteriophage, yeast episomes, etc.), yeast chromosomal elements, viruses (vaculovirus, etc.).
  • examples include papovavirus, vaccinia virus, adenovirus, adeno-associated virus, tripoxvirus, pseudomad dog disease virus, herpesvirus, lentivirus, retrovirus, etc.), and vectors derived from these combinations (cosmid, phagemid, etc.).
  • the plasmid vector is preferable because of its high versatility.
  • a plasmid vector is preferable in that a large amount of donor DNA can be introduced into the cell.
  • the vector contains a splicing acceptor sequence (SA sequence) on the 5'end side of a polynucleotide sequence in which the translation initiation codon is replaced with a nontranslation initiation codon.
  • the vector has a cleavage site (such as a gRNA recognition sequence), a buffering polynucleotide, and a splicing acceptor sequence (such as a gRNA recognition sequence) on the 5'end of a polynucleotide sequence in which the translation initiation codon has been replaced with a non-translation initiation codon.
  • SA sequence is included in this order.
  • the composition of the vector is such that a cleavage site (gRNA recognition sequence, etc.), a polynucleotide for buffering, and a splicing acceptor sequence are arranged on the 5'terminal side of the modified polynucleotide 1a.
  • the splicing acceptor sequence When producing the above-mentioned mature mRNA without using the splicing acceptor sequence, it is necessary to knock in the modified polynucleotide 1a immediately after exon E2. On the other hand, if the splicing acceptor sequence is used, it is not necessary to knock in the modified polynucleotide 1a immediately after the exon E2. Instead, the modified polynucleotide 1a may be knocked in anywhere in the intron I2. Therefore, the use of the splicing acceptor sequence has an advantage that the number of knock-in sites of the modified polynucleotide 1a can be increased. This advantage is, in other words, the advantage of facilitating the design of gRNAs.
  • indel may occur at the boundary region between the polynucleotide 1a and the exon E2. If indel occurs at the boundary region between polynucleotide 1a and exon E2, the amino acid sequence encoded by exon E2 may change, or a frame shift may occur during translation of polynucleotide 1a.
  • the insertion site should be intron I1 (intron between the first exon and the second exon). It is preferable to set it.
  • the SA sequence may be a sequence unique to the knock-in site or a more versatile sequence (see Examples 4 and 5 for details).
  • the vector according to one embodiment of the present invention may contain an arbitrary sequence in addition to the modified polynucleotide 1a.
  • the modified polynucleotide 1a may contain a polynucleotide sequence that expresses another protein (such as Cas9).
  • kit ⁇ One aspect of the present invention is a genetically modified kit comprising the above-mentioned polynucleotide or the above-mentioned vector.
  • kit means any combination of reagents and the like used for any purpose. This use may be for medical use or for experimental use. Specifically, the application may be a genome editing application.
  • the kit comprises reagents and / or ancillary substances used for genetic recombination.
  • reagents and / or ancillary substances include nucleases (such as Cas), gRNAs, TALENs, ZFNs.
  • the kit may include one or more containment vessels (boxes, bottles, dishes, etc.) for storing reagents and / or ancillary substances.
  • the kit may include two or more containers, each container containing a portion of the components of the kit. At this time, the user may receive each container collectively or individually.
  • Protein (modified recombinant enzyme) One aspect of the present invention is a protein (modified recombinant enzyme) which is a translation product of the above-mentioned polynucleotide (modified polynucleotide 1a).
  • modified polynucleotide 1a modified polynucleotide 1a
  • the protein is a recombinant enzyme consisting of the amino acid sequence set forth in SEQ ID NO: 10, 11 or 12.
  • These recombinant enzymes are translation products of "Opti Cre2", “Opti FLP” and “Opti Dre” in Examples described later.
  • the protein may be a fusion protein with another protein. Since the method for producing the fusion protein is well known in the art, the description thereof will be omitted.
  • the genetic recombination method includes the step of introducing the above-mentioned polynucleotide or the above-mentioned vector into a cell or a subject (for example, a human subject or a non-human subject).
  • introducing into a cell is intended to introduce, for example, a polynucleotide or vector into a cell in vitro.
  • introducing into a subject is intended to introduce, for example, a polynucleotide or vector into cells constituting a living body in vivo.
  • the subject is not human.
  • the subject is a non-human mammal.
  • non-human mammals include cloven-hoofed animals (boars, wild boars, pigs, sheep, goats, etc.), cloven-hoofed animals (horses, etc.), carnivores (mouse, rats, hamsters, squirrels, etc.) Etc.), meats (dogs, cats, ferrets, etc.).
  • the non-human mammals described above include wild animals in addition to livestock or companion animals (pets).
  • the method for introducing a polynucleotide or vector into a cell or a subject is not particularly limited.
  • a method for introducing a vector into a cell or a subject an appropriate method depending on the vector is widely known in the art.
  • Methods for introducing a polynucleotide as a DNA molecule (or RNA molecule) into a cell or subject include electroporation, microinjection, sonoporation, laser irradiation, and cationic substances (cationic polymers, cationic lipids, etc.). Transfection using complexation with calcium phosphate, etc.) can be mentioned.
  • the gene recombination method according to one embodiment of the present invention is preferably combined with an introduction form that introduces a large amount of unmodified DNA into a cell.
  • introduction forms include electroporation, virus, microinjection, lipid complex transfection and the like.
  • the protein encoded by the modified polynucleotide 1a is a Cre modified protein.
  • the modified polynucleotide 1a has one or more of the wild-type Cre translation initiation codons ATG replaced with non-translation initiation codons (preferably leucine-encoding codons). More preferably, the modified polynucleotide 1a contains one or more of the translation initiation codons (ATGs) corresponding to the 1st, 28th, 30th, 58th and 77th methionine residues of wild Cre. , Is replaced with an untranslated start codon (preferably a codon encoding leucine).
  • ATGs translation initiation codons
  • the modified polynucleotide 1a has a translation initiation codon (ATG) corresponding to the 1st, 28th, 30th, 58th and 77th methionine residues of wild-type Cre, which is a non-translation initiation codon (ATG). Preferably, it is substituted with a codon encoding leucine).
  • ATG translation initiation codon
  • ATG non-translation initiation codon
  • ATG non-translation initiation codon
  • it is substituted with a codon encoding leucine.
  • An example of the base sequence of such a modified polynucleotide 1a is the base sequence shown in SEQ ID NO: 5. This base sequence corresponds to "Opti Cre" in the examples.
  • the modified polynucleotide 1a is one in which one or more of the atypical translation initiation codons of wild Cre is substituted with the untranslation initiation codon in addition to the substitution of the translation initiation codon ATG described above. is there.
  • the atypical translation start codon to non-translation start codon substitutions are (i) CTG to CTC substitution, (ii) TTG to TTA substitution, (iii) GTG to GTA substitution, Or (iv) substitution from ATA to ATT.
  • the atypical translation initiation codon substituted for the untranslation initiation codon is within 30 bp, within 60 bp, within 90 bp, within 120 bp, within 180 bp, 210 bp from the 5'end of the polynucleotide encoding the wild-type Cre.
  • An example of the base sequence of such a modified polynucleotide 1a is the base sequence shown in SEQ ID NO: 6. This base sequence corresponds to "Opti Cre2" in the examples.
  • Cre-modified protein which is a translation product of the modified polynucleotide 1a of the embodiment described in this item, is also a preferred embodiment of the present invention.
  • amino acid sequence of the Cre modified protein include the amino acid sequence set forth in SEQ ID NO: 10. This amino acid sequence is a translation of "Opti Cre” or "Opti Cre 2" in the Examples. It has been suggested that this translation product may have reduced toxicity found in wild-type Cre (see Example 7 for details).
  • pLeaklessIII vector developed in 2016 development
  • Infusion manufactured by TAKARA
  • TAKARA TAKARA
  • Recombinant enzyme Wild-type Cre, Cre (Opti Cre) in which the translation start codon (ATG) was replaced with the untranslated start codon, and Cre (Opti Cre2) in which the atypical translation start codon was also replaced with the untranslated start codon were used.
  • the codon (ATG) corresponding to the 1st, 28th, 30th, 58th, and 77th methionine residues of wild-type Cre is encoded by the leucine residue. Substituted with codons (TTA, TTG, CTT, CTC, CTA or CTG).
  • Opti Cre2 the atypical translation initiation codon contained in Opti Cre was also replaced with the non-translation initiation codon. Specifically, CTG, which exists from the 5'end to 600 bp, was replaced with CTC, TTG was replaced with TTA, GTG was replaced with GTA, and ATA was replaced with ATT.
  • CTC which exists from the 5'end to 600 bp
  • TTG was replaced with TTA
  • GTG was replaced with GTA
  • ATA was replaced with ATT.
  • the amino acid encoded by the codon before substitution and the amino acid encoded by the codon after substitution are the same amino acid.
  • the nucleotide sequence encoding the wild-type Cre is SEQ ID NO: 1, and the amino acid sequence of the wild-type Cre is SEQ ID NO: 9.
  • the nucleotide sequence encoding Opti Cre is SEQ ID NO: 5, and the amino acid sequence of Opti Cre is SEQ ID NO: 10.
  • the nucleotide sequence encoding Opti Cre2 is the same as SEQ ID NO: 6, and the amino acid sequence of Opti Cre2 is the same as the amino acid sequence of Opti Cre.
  • Humanized Cas9 (hCas9) was used for the introduction of the recombinant enzyme.
  • the nucleotide sequence encoding hCas9 is SEQ ID NO: 13.
  • mCherry red fluorescent protein
  • flox-STOP-EGFP sequence a sequence constructed so that EGFP is expressed by the expression of Cre
  • the base sequence encoding mChery is SEQ ID NO: 14.
  • the nucleotide sequence encoding the flox-STOP-EGFP sequence is SEQ ID NO: 15.
  • the CAG promoter was used as the promoter sequence for non-specific gene expression.
  • a CAX promoter sequence modified from the CAG promoter was used as a promoter sequence for site-specific expression of a gene in a nerve cell.
  • the promoter regions of the CAG promoter and the CAX promoter are the same, and the SEQ ID NO: 24.
  • SA sequence The SA sequence of mouse Tubb3 gene 4th exon (SEQ ID NO: 16) or the SA sequence of human immunoglobulin (chimeric intron; SEQ ID NO: 17) was used.
  • the plasmid was introduced into the cerebral cortex of a mouse (or ferret) fetal by in utero electroporation.
  • Example 1 Leakage of wild-type Cre gene
  • the presence or absence of leaks from the donor DNA was examined using donor DNA containing wild-type Cre (Cr that did not replace the translation initiation codon). Specifically, wild-type Cre was knocked in to the Tubb3 locus specifically expressed in nerve cells. The HITI method was adopted for gene transfer. Since the HITI method does not require a sequence such as a homology arm, the influence of sequences other than Cre on the leak can be minimized.
  • the specific composition of the donor DNA containing the wild-type Cre is shown in FIG.
  • a gRNA recognition sequence SEQ ID NO: 18
  • SEQ ID NO: 18 a gRNA recognition sequence existing at the 3'end of the Tubb3 gene is arranged at both ends of the base sequence encoding the wild-type Cre.
  • mice introduced with an expression vector other than the hCas9 expression vector were used as negative controls.
  • -PCAX-hCas9 expression vector of hCas9
  • PCAG-mCherry-gRNA expression vector of mCherry and gRNA
  • PCAG-flox-STOP-EGFP reporter vector containing the fluorx-STOP-EGFP sequence
  • Cre Donner DNA
  • Example 2 Relationship between methionine contained in wild-type Cre and recombinant activity
  • the following vector was introduced into the cerebral cortex of a mouse fetal on the 14th day of embryonic development.
  • -PCAG-Cre-1-6 expression vector of any of Cre1 to Cre6
  • PCAG-mChery expression vector of mCherry
  • PCAG-flox-STOP-EGFP reporter vector containing the fluorx-STOP-EGFP sequence.
  • Example 3 Leakage of Opti Cre gene
  • Opti Cre was introduced into the intron and expressed using donor DNA containing the splicing acceptor sequence (SA sequence) and Opti Cre. Specifically, a donor vector in which the SA sequence (SA sequence of the 4th exon of the mouse Tubb3 gene), the base sequence encoding Opti Cre, and the 3'UTR region of the mouse Tubb3 gene are arranged in order from the 5'end side. (See the upper panel in FIG. 8 for a more detailed configuration). This donor DNA was introduced into the intron region of the mouse Tubb3 gene by the HITI method (see the lower panel of FIG. 8).
  • the gRNA recognition sequence used is SEQ ID NO: 19.
  • mice introduced with an expression vector other than the hCas9 expression vector were used as negative controls.
  • -PCAX-hCas9 expression vector of hCas9
  • PgRNA expression vector of gRNA
  • PCAG-mCherry expression vector of mCherry
  • PCAG-flox-STOP-EGFP reporter vector containing the fluorx-STOP-EGFP sequence
  • PCAG hyper piggybase expression vector of hyper piggybase
  • Donner DNA containing SA sequence and Opti Cre.
  • the brain was excised to prepare a section of the cerebral cortex. This section was antibody-stained with an antibody against mCherry and an antibody against EGFP and observed under a microscope. The result is shown in FIG.
  • Opti Cre can be expressed site-specifically by the gene trap method when a donor DNA in which the SA sequence is arranged on the 5'terminal side of Opti Cre is used.
  • Example 5 Donor DNA having SA sequence (No. 2)
  • an endogenous SA sequence unique to the target site for gene transfer was used.
  • Opti Cre was used with a more versatile endogenous SA sequence.
  • the SA sequence (chimeric intron) of human immunoglobulin was used as the SA sequence (see FIG. 10 for the specific composition of the donor DNA). The experiment was carried out in the same manner as in Example 4 except that the SA sequence was changed. The results are shown in FIG.
  • EGFP expression was observed in the hCas9-introduced group, whereas EGFP expression was not observed in the hCas9-introduced group. That is, in the cells in which knock-in did not occur, leakage from the donor DNA was suppressed. This suggests that Opti Cre can be expressed site-specifically by the gene trap method even when a non-endogenous SA sequence is used.
  • Example 6 Application to a non-mouse model and a model using HDR
  • HDR Homology Directed Repair
  • Example 6 a modified Cre (Opti Cre2) in which the atypical translation start codon was substituted in addition to ATG was used.
  • the target of gene transfer was the Pax6 gene specifically expressed in undifferentiated cells (the gRNA recognition sequence used was SEQ ID NO: 20).
  • a vector containing mCherry and flox-STOP-EGFP sequences contained LR sequences (SEQ ID NOs: 21 and 22), and
  • hyper piggybase which is a type of transposon, was also introduced into the genome. This allows the mCherry and flox-STOP-EGFP sequences to be randomly integrated into the genome. Therefore, it is possible to prevent the proportion of cells incorporating the mCherry and flox-STOP-EGFP sequences from being reduced by cell division.
  • the following vector was introduced into the cerebral cortex of a ferret fetal on the 30th day of embryonic development. Further, a ferret into which an expression vector other than the expression vector of hCas9 was introduced was used as a negative control.
  • -PCAX-hCas9 expression vector of hCas9
  • -PLR5 CAG-mCherry mCherry expression vector; has LR sequence
  • gRNA expression vector gRNA expression vector
  • PLR5 CAG-flox-STOP-EGFP reporter vector containing fluorx-STOP-EGFP sequence; having LR sequence
  • HDR Cre Donor Donator DNA of Opti Cre2
  • PCAX hyper piggybase expression vector of hyper piggybase
  • Example 7 Comparison of activity between OptiCre2 and wild-type Cre.
  • the recombinant activity of OptiCre2 was compared with the recombinant activity of wild-type Cre by an assay using cultured cells.
  • 293T cells were seeded on a 6-well plate and the following vector was introduced using lipofectamine 2000.
  • -PCAG-Cre or pCAG optiCre2 Expression vector of Cre or OptiCre2, respectively
  • -CAG-flox-STOP-EGFP reporter vector containing the fluorx-STOP-EGFP sequence
  • ATG was placed on the 5'end side of the base sequence encoding OptiCre2 so that OptiCre2 could be translated normally.
  • pCAG-Cre or pCAG optiCre2 was made into a dilution series and injected into wells at gradual concentrations (1 ⁇ g / well, 100 ng / well, 10 ng / well, 1 ng / well, and 10 pg / well).
  • the ratio of EGFP-expressing cells to all cells was measured by FACS. This measurement was standardized by dividing by the percentage of all cells expressing EGFP in positive control.
  • a positive control cells into which pCAG-EGFP (EGFP introduction vector) was introduced were used (1 ⁇ g / well).
  • a negative control untransfected cells were used.
  • the measurement results by FACS are shown in FIGS. 14 to 17, and the results after standardization are shown in FIGS.
  • OptiCre2 has a recombination activity equivalent to that of wild-type Cre.
  • Opti Cre2 may reduce the toxicity of wild-type Cre. That is, it was suggested that Opti Cre2 is a variant that not only reduces the leakage from the donor DNA but also reduces the toxicity of the wild-type Cre.
  • Example 8 Modified activity of FLP and Dre
  • variants of FLP and Dre which are enzymes frequently used other than Cre, were prepared and their activities were examined. Specifically, similarly to OptiCre2, a variant in which the translation initiation codon contained in the wild-type FLP and the wild-type Dre was replaced with the non-translation initiation codon was prepared. These variants are referred to as Opti FLP and Opti Dre, respectively.
  • the specific sequence is SEQ ID NO: 7 for the base sequence of Opti FLP, SEQ ID NO: 11 for the amino acid sequence of Opti FLP, SEQ ID NO: 8 for the base sequence of Opti Dre, and SEQ ID NO: 12 for the amino acid sequence of Opti Dre.
  • the present invention can be used, for example, in the field of biotechnology (for example, genome editing).
  • Polynucleotide encoding a recombinant enzyme 1a Modified polynucleotide (polynucleotide according to one embodiment of the present invention) 5: Translation start codon 5a: Non-translation start codon

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Abstract

Le but d'un mode de réalisation de la présente invention est de fournir un polynucléotide pouvant réduire les pertes. Le polynucléotide (le polynucléotide modifié 1a) selon un mode de réalisation de la présente invention : (I) est un polynucléotide (1a) étant produit en remplaçant au moins un codon d'initiation de traduction (5) d'un polynucléotide (1) codant pour une enzyme de recombinaison (ou une variante du polynucléotide (1) codant pour une enzyme de recombinaison) avec un codon d'initiation de non-traduction (5a) ; et (ii) des codes pour une protéine ayant une activité de recombinaison.
PCT/JP2020/040738 2019-10-31 2020-10-30 Polynucléotide et son utilisation Ceased WO2021085580A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003070931A2 (fr) * 2002-02-21 2003-08-28 Vision 7 Gmbh Techniques de recombinaison d'adn a des sites specifiques
US20140283156A1 (en) * 2013-03-14 2014-09-18 Cold Spring Harbor Laboratory Trans-splicing ribozymes and silent recombinases
JP2017526341A (ja) * 2014-09-02 2017-09-14 ハインリヒ−ペッテ−インスティトゥート・ライブニッツ−インスティトゥート・フューア・エクスペリメンテレ・ヴィロロギー − シュティフトゥング・ビュルガーリヒェン・レッヒツＨｅｉｎｒｉｃｈ−Ｐｅｔｔｅ−Ｉｎｓｔｉｔｕｔ Ｌｅｉｂｎｉｚ−Ｉｎｓｔｉｔｕｔ Ｆｕｒ Ｅｘｐｅｒｉｍｅｎｔｅｌｌｅ Ｖｉｒｏｌｏｇｉｅ − Ｓｔｉｆｔｕｎｇ Ｂｕｒｇｅｒｌｉｃｈｅｎ Ｒｅｃｈｔｓ 複数のレトロウイルス株における非対称標的部位を組み換えるための、十分に寛容化され高度に特異的なテーラーメイドリコンビナーゼ
JP2018500037A (ja) * 2014-12-31 2018-01-11 シンセティック ジェノミクス インコーポレーテッド 高効率なインビボゲノム編集のための組成物及び方法
JP2018506293A (ja) * 2015-02-24 2018-03-08 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 結合誘発型転写スイッチ及びその使用方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003070931A2 (fr) * 2002-02-21 2003-08-28 Vision 7 Gmbh Techniques de recombinaison d'adn a des sites specifiques
US20140283156A1 (en) * 2013-03-14 2014-09-18 Cold Spring Harbor Laboratory Trans-splicing ribozymes and silent recombinases
JP2017526341A (ja) * 2014-09-02 2017-09-14 ハインリヒ−ペッテ−インスティトゥート・ライブニッツ−インスティトゥート・フューア・エクスペリメンテレ・ヴィロロギー − シュティフトゥング・ビュルガーリヒェン・レッヒツＨｅｉｎｒｉｃｈ−Ｐｅｔｔｅ−Ｉｎｓｔｉｔｕｔ Ｌｅｉｂｎｉｚ−Ｉｎｓｔｉｔｕｔ Ｆｕｒ Ｅｘｐｅｒｉｍｅｎｔｅｌｌｅ Ｖｉｒｏｌｏｇｉｅ − Ｓｔｉｆｔｕｎｇ Ｂｕｒｇｅｒｌｉｃｈｅｎ Ｒｅｃｈｔｓ 複数のレトロウイルス株における非対称標的部位を組み換えるための、十分に寛容化され高度に特異的なテーラーメイドリコンビナーゼ
JP2018500037A (ja) * 2014-12-31 2018-01-11 シンセティック ジェノミクス インコーポレーテッド 高効率なインビボゲノム編集のための組成物及び方法
JP2018506293A (ja) * 2015-02-24 2018-03-08 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 結合誘発型転写スイッチ及びその使用方法

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