WO2024063273A1 - Nouveaux variants d'adénine désaminase et procédé d'édition de bases les utilisant - Google Patents

Nouveaux variants d'adénine désaminase et procédé d'édition de bases les utilisant Download PDF

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WO2024063273A1
WO2024063273A1 PCT/KR2023/009361 KR2023009361W WO2024063273A1 WO 2024063273 A1 WO2024063273 A1 WO 2024063273A1 KR 2023009361 W KR2023009361 W KR 2023009361W WO 2024063273 A1 WO2024063273 A1 WO 2024063273A1
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dna
fusion protein
target
editing
amino acid
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Jin-Soo Kim
Sung Ik Cho
Kayeong LIM
Jaesuk Lee
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Institute For Basic Science
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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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 RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04002—Adenine deaminase (3.5.4.2)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor

Definitions

  • an adenine deaminase variant capable of reducing off-target editing
  • a fusion protein comprising a DNA-binding protein, and the adenine deaminase variant
  • a base editing composition for A-to-G base editing in DNA comprising the fusion protein
  • a method for A-to-G base editing in DNA comprising delivering the base editing composition to a cell containing a target DNA.
  • mtDNA mammalian mitochondrial DNA
  • Programmable deaminases which are composed of a custom DNA-binding protein and a nucleobase deaminase, enable mitochondrial DNA editing in a targeted manner.
  • adenine base editors In contrast to cytosine base editors, adenine base editors, known as TALEDs, incorporate TadA8e, a deoxy-adenine deaminase engineered from the tRNA-specific TadA protein derived from E. coli. TadA8e and related variants of TadA are crucial components in CRISPR RNA-guided adenine base editors, extensively employed for A-to-G base editing of nuclear DNA. Nevertheless, the use of RNA-guided ABEs for editing organellar DNA poses a challenge due to the difficulty of delivering guide RNA into organelles.
  • TadA8e present in ABEs retains residual deaminase activity for RNA substrates, resulting in unintended, transcriptome-wide off-target base editing. Off-target effects arise when the editing machinery mistakenly acts on unintended genomic regions, leading to undesired modifications.
  • the present invention relates to a base editing composition and its method of application in gene therapy and genome engineering. More specifically, the composition and method involve base editor variants that exhibit a significant reduction in off-target genome cleavage and side effects, such as RNA off-target toxicity, when employed in clinical settings.
  • the present invention offers a fresh avenue for targeted base editing with wide-ranging applications in the fields of medicine and biotechnology.
  • an adenine deaminase variant in one embodiment, provided herein is an adenine deaminase variant.
  • a fusion protein comprising a DNA-binding protein, and the adenine deaminase variant.
  • a polynucleotide encoding the adenine deaminase variant or the fusion protein, and an expression vector comprising the polynucleotide.
  • a base editing composition for A-to-G base editing in DNA comprising the fusion protein, a polynucleotide encoding the fusion protein or an expression vector comprising the polynucleotide.
  • a method for A-to-G base editing in DNA comprising delivering the base editing composition to a cell containing a target DNA.
  • described herein is a method for reducing off-target editing effects, the method comprising delivering the base editing composition to a cell containing a target DNA.
  • adenine deaminase variant or the fusion protein in A-to-G base editing in DNA and/or reducing off-target effect in A-to-G base editing in DNA, or in preparing a composition for A-to-G base editing in DNA and/or reducing off-target effect in A-to-G base editing in DNA.
  • FIG. 1(a) exemplifies the structures of base editors used in the present invention.
  • AD TedA8e adenine deaminase
  • AD* TedA8e adenine deaminase variant
  • MTS mitochondrial targeting sequence
  • UGI uracil glycosylase inhibitor
  • FIG. 1(b) is a graph showing the number of RNA edits and editing frequencies for the Cox3.1-specific sTALEDs.
  • FIG. 1(c) is graph showing A-to-G edits and C-to-T edits for the Cox3.1-specific sTALEDs (left), as well as on-target activity of Cox3.1-specific sTALED variants relative to that of the wild-type Cox3.1-specific sTALED (right).
  • FIG. 1(d) is a graph showing the number of RNA edits and editing frequencies for the ND1-specific sTALEDs.
  • FIG. 1(e) is graphs showing A-to-G edits and C-to-T edits for the ND1-specific sTALEDs (left), as well as on-target activity of the ND1-specific sTALED variants relative to that of the wild-type ND1-specific sTALED (right).
  • FIG. 2(a) Structural representations of the TadA portion of ABE8e (Protein Data Bank (PDB) accession number 6VPC).
  • FIG. 2(b) is a heat map showing DNA on-target activity (left), RNA off-target activity (middle), and the relative ratio of DNA on-target editing frequencies to RNA off-target editing frequencies (right) of the 101 sTALED variants among the total of 209 sTALED variants that retain mitochondrial DNA on-target activity.
  • the relative ratio is normalized to that for the original sTALED, which has a value of 1.
  • FIG. 3 is graphs showing DNA on-target base editing frequencies induced by 209 sTALED variants.
  • FIG. 4(a) is a graph showing the editing frequencies at six RNA off-target sites measured by targeted RNA sequencing.
  • FIG. 4(b) is a graph showing RNA off-target editing frequencies induced by sTALED and sTALED variants extracted from transcriptome-wide sequencing at six selected RNA off-target sites.
  • FIG. 4(c) is a graph showing RNA off-target editing frequencies induced by sTALED and sTALED variants analyzed by targeted RNA amplicon sequencing.
  • FIG. 5(a) is a graph showing the number of RNA edits and editing frequencies for the Cox3.1-specific sTALEDs.
  • FIG. 5(b) is graphs showing A-to-G edits and C-to-T edits for the Cox3.1-specific sTALEDs.
  • FIG. 5(c) is a graph showing the number of RNA edits and editing frequencies for the ND1-specific sTALEDs.
  • FIG. 5(d) is graphs showing A-to-G edits and C-to-T edits for the ND1-specific sTALEDs.
  • FIG. 5(e) is a graph showing the number of RNA edits and editing frequencies for the ND6-specific sTALEDs.
  • FIG. 5(f) is a graph showing A-to-G edits and C-to-T edits for the ND6-specific sTALEDs.
  • FIG. 6(a) is a graph showing on-target activities of ND1-specific sTALED variants relative to that of wild-type ND1-specific sTALED.
  • FIG. 6(b) is a graph showing on-target activities of ND6-specific sTALED variants relative to that of wild-type ND6-specific sTALED.
  • FIG. 6(c) is a heat map showing RNA off-target activities of ND1-specific sTALED and sTALED variants at six representative sites.
  • FIG. 6(d) is a heat map showing RNA off-target activities of ND6-specific sTALED and sTALED variants at six representative sites.
  • FIG. 6(g) is a bar graph showing the ratio of DNA on-target editing frequencies relative to RNA off-target editing frequencies induced by ND1-specific sTALED and sTALED variants.
  • FIG. 6(h) is a bar graph showing the ratio of DNA on-target editing frequencies relative to RNA off-target editing frequencies induced by ND6-specific sTALED and sTALED variants. This ratio is normalized to that for sTALED, which has a value of 1.
  • FIG. 6(i) is a graph showing average relative ratio values for the sTALEDs and sTALED variants targeted to Cox3.1, ND1 (FIG. 6(g)), and ND6 (FIG. 6(h)).
  • FIG. 7(a) is a heat maps depicting A-to-G conversions caused by sTALED or sTALED variants targeted to the Cox3.1 site.
  • FIG. 7(b) is a heat maps depicting A-to-G conversions caused by sTALED or sTALED variants targeted to the ND1 site.
  • FIG. 7(c) is a heat maps depicting A-to-G conversions caused by sTALED or sTALED variants targeted to the ND6 site.
  • FIGs. 7(d)-(f) show the analysis of Cox3.1, ND1, ND6 alleles summarized in FIGs. 7(a)-(c), respectively.
  • the spacer sequence is shown on the left, and bar graphs displaying the frequency of each allele are shown on the right.
  • the reference sequence is written all in capital letters, whereas the lowercase letters indicate the positions at which base editing has taken place.
  • FIG. 8(a) shows the plots indicating the positions of on-target and off-target edits across the mitochondrial genome at day 4 post-transfection.
  • Black, and gray dots indicate off-target edits, and naturally-occurring single-nucleotide variations (SNVs), respectively, and the arrows indicate on-target (and bystander) edits.
  • Nucleotide positions in the human mitochondrial genome are represented on the X axis.
  • FIG. 8(b) shows the average frequencies of genome-wide off-target edits induced by wild-type TALED and TALED variants.
  • FIG. 9(a) shows the plots indicating the positions of on-target and off-target edits across the mitochondrial genome at day 2 post-transfection.
  • Black, and gray dots indicate off-target edits, and naturally-occurring single-nucleotide variations (SNVs), respectively, and the arrows indicate on-target (and bystander) edits.
  • Nucleotide positions in the human mitochondrial genome are represented on the X axis.
  • FIG. 11(a) illustrated an experimental scheme of one embodiment of the present invention.
  • FIGs. 11(b) and (c) are bar graphs showing viability of cells transfected with plasmids expressing sTALED, sTALED-V106W, sTALED-V28R and sTALED-R111S targeted to the indicated sites which was determined by observing the color change caused by formazan formation in an MTS assay at day 2 (B) and day 4 (C) post-transfection.
  • FIG. 12(a) exemplifies the architecture of ABE8e and ABE8e variant constructs.
  • AD TedA8e adenine deaminase
  • AD* TedA8e adenine deaminase variant
  • NLS nuclear localization sequence
  • FIG. 12(b) is a graph showing on-target activity of ABE8e and the ABE8e variants (ABE8e-V106W, ABE8e-V28R, ABE8e-R111S) at the nuclear TYRO3 site.
  • FIG. 12(c) depicts a heat map showing the frequencies of A-to-G conversions caused by ABE8e and the ABE8e variants (ABE8e-V106W, ABE8e-V28R, ABE8e-R111S) at the nuclear TYRO3 site.
  • FIG. 12(d) is a graph showing RNA off-target activity of TYRO3-targeted ABE8e and ABE8e variants (ABE8e-V106W, ABE8e-V28R, ABE8e-R111S) at six representative sites.
  • FIGs. 12(e) and 12(f) are graphs illustrating the total number of RNA edits found in HEK 293T cells that expressed ABE8e or the ABE8e variants targeted to the nuclear TYRO3 site as assessed by whole transcriptome sequencing.
  • FIG. 13(a) is a graph showing DNA on-target activity of Cox3-specific TALEDs including dimeric TALEDs (dTALEDs), half monomer (dTALED-ADs), monomeric TALEDS (mTALEDs), and untreated samples.
  • dTALEDs dimeric TALEDs
  • dTALED-ADs half monomer
  • mTALEDs monomeric TALEDS
  • FIG. 13(b) is a graph showing RNA off-target activity of Cox3-specific TALEDs including dimeric TALEDs (dTALEDs), half monomer (dTALED-ADs), monomeric TALEDS (mTALEDs), and untreated samples at six representative sites.
  • dTALEDs dimeric TALEDs
  • dTALED-ADs half monomer
  • mTALEDs monomeric TALEDS
  • FIG. 13(c) is a graph showing specificity ratios of RNA off-target editing relative to on-target editing induced by Cox3-specific TALEDs.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, such as within 5-fold or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • corresponding refers to amino acid residues at positions listed in the polypeptide or amino acid residues that are similar, identical, or homologous to those listed in the polypeptide. Identifying the amino acid at the corresponding position may be determining a specific amino acid in a sequence that refers to a specific sequence.
  • corresponding region generally refers to a similar or corresponding position in a related protein or a reference protein. For example, an arbitrary amino acid sequence is aligned with SEQ ID NO: 3, and based on this, each amino acid residue of the amino acid sequence may be numbered with reference to the amino acid residue of SEQ ID NO: 3 and the numerical position of the corresponding amino acid residue.
  • a sequence alignment algorithm as described in the present disclosure may determine the position of an amino acid or a position at which modification such as substitution, insertion, or deletion occurs through comparison with that in a query sequence (also referred to as a "reference sequence").
  • alignment means mapping sequence reads to a reference genome and then aligning the bases having identical sites in genomes to fit for each site. Accordingly, so long as it can align sequence reads in the same manner as above, any computer program may be employed.
  • the program may be one already known in the pertinent art or may be selected from among programs tailored to the purpose. In one embodiment, alignment is performed using ISAAC, but is not limited thereto.
  • the term “host cell” (or “recombinant host cell”), as used herein, is intended to refer to a cell that has been genetically altered, or is capable of being genetically altered by introduction of an exogenous polynucleotide molecule, such as a recombinant plasmid or vector. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progency may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.
  • the expression "base editor (BE)” is meant an agent that binds a polynucleotide and has nucleobase modifying activity.
  • the base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain.
  • the base editor comprises a nucleobase modifying polypeptide (e.g., a deaminase) and a nucleic acid programmable nucleotide binding domain in conjunction with a guide polynucleotide (e.g., guide RNA).
  • the agent is a biomolecular complex comprising a protein domain having base editing activity, i.e., a domain capable of modifying a base (e.g., A, T, C, G, I, or U) within a nucleic acid molecule (e.g., DNA).
  • a protein domain having base editing activity i.e., a domain capable of modifying a base (e.g., A, T, C, G, I, or U) within a nucleic acid molecule (e.g., DNA).
  • the polynucleotide programmable DNA binding domain is fused or linked to a deaminase domain.
  • the agent is a fusion protein comprising a domain having base editing activity.
  • the protein domain having base editing activity is linked to the guide RNA (e.g., via an RNA binding motif on the guide RNA and an RNA binding domain fused to the deaminase).
  • the domain having base editing activity is capable of deaminating a base within a nucleic acid molecule.
  • the base editor is capable of deaminating one or more bases within a DNA molecule.
  • the base editor is capable of deaminating an adenine (A) within DNA.
  • the base editor is an adenine base editor (ABE).
  • composition administration e.g., injection
  • composition administration can be performed by intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, or intramuscular (i.m.) injection.
  • s.c. sub-cutaneous injection
  • i.d. intradermal
  • i.p. intraperitoneal
  • intramuscular injection i.m.
  • Parenteral administration can be, for example, by bolus injection or by gradual perfusion over time.
  • parenteral administration includes infusing or injecting intravascularly, intravenously, intramuscularly, intraarterially, intrathecally, intratumorally, intradermally, intraperitoneally, transtracheally, subcutaneously, subcuticularly, intraarticularly, subcapsularly, subarachnoidly and intrasternally.
  • administration can be by the oral route.
  • another amino acid may be intended to refer to an amino acid selected from among alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, asparagine, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, lysine, and all known variants thereof, exclusive of the amino acid having a wild-type protein retained at the original substitution position.
  • off-target site may refer to a site that is not an on-target site, but to which the adenine base editors show activity. That is, the off-target site may refer to a site where base editing occurs, besides an on-target site.
  • the term "off-target site” may be used to cover not only sites that are not on-target sites of the adenine base editors, but also sites having possibility to be off-target sites thereof.
  • the term “whole genome sequencing” refers to a method of reading the genome by many multiples such as in 10X, 20X, and 40X formats for whole genome sequencing by next generation sequencing.
  • Next generation sequencing means a technology that fragments the whole genome or targeted regions of genome in a chip-based and PCR-based paired end format and performs sequencing of the fragments by high throughput on the basis of chemical reaction (hybridization).
  • nucleic acid refers to either DNA or RNA.
  • Nucleic acid sequence or “polynucleotide sequence” refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA, and nonfunctional DNA or RNA.
  • nucleic acid molecule encoding refers to a nucleic acid molecule which directs the expression of a specific protein or peptide.
  • the nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein or peptide.
  • the nucleic acid molecule includes both the full-length nucleic acid sequences as well as non-full length sequences derived from the full length protein. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference in a specific host cell.
  • vector refers to viral expression systems, autonomous self-replicating circular DNA (plasmids), and includes both expression and nonexpression plasmids. Where a recombinant microorganism or cell is described as hosting an “expression vector,” this includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome(s). Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or the vector may be incorporated within the host's genome.
  • plasmid refers to an autonomous circular DNA molecule capable of replication in a cell, and includes both the expression and nonexpression types. Where a recombinant microorganism or cell is described as hosting an “expression plasmid”, this includes latent viral DNA integrated into the host chromosome(s). Where a plasmid is being maintained by a host cell, the plasmid is either being stably replicated by the cell during mitosis as an autonomous structure, or the plasmid is incorporated within the host's genome.
  • the “percentage amino acid sequence homology” or percent amino acid sequence identity” refers to between a first amino acid sequence and a second amino acid sequence. may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence-compared to the first amino acid sequence-is considered as a difference at a single amino acid residue (position), i.e. as an “amino acid difference” as defined herein.
  • the degree of sequence identity between two amino acid sequences may be calculated using a known computer algorithm, such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings.
  • RNA off-target effects include, for example, bystander off-target effects
  • DNA off-target effects including, for example, bystander off-target effects
  • such objective was accomplished by substituting different amino acid residues at specific locations within TadA8e that interact with nucleotides.
  • RNA sequencing as well as whole mitochondrial genome sequencing were performed. This comprehensive analysis allowed for the identification and confirmation of either the entire spectrum of RNA off-target sites or a representative selection of six prominent RNA off-target sites.
  • the dynamics of RNA off-target effects over time were also measured. Such measurements were taken at various time points to assess how the RNA off-target landscape changed over the course of expression.
  • an adenine deaminase comprising the amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 80% sequence homology to the amino acid sequence of SEQ ID NO:1, wherein at least one amino acid residue selected from residues 28, 30, 46, 48, 49, 82, 84, 106, 108, 110, and 111 of SEQ ID NO:1 or the corresponding amino acid residue of the amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence homology (or sequence identity) to the amino acid sequence of SEQ ID NO:1 is substituted with another amino acid.
  • adenine deaminase refers to a polypeptide or a fragment capable of catalyzing the hydrolytic deamination of adenine or adenosine.
  • the deaminase or deaminase domain represents an adenine deaminase that facilitates the hydrolytic deamination of adenosine to inosine or deoxyadenosine to deoxyinosine.
  • the adenine deaminase performs the hydrolytic deamination of adenine or adenosine in DNA (deoxyribonucleic acid).
  • the adenosine deaminases such as engineered adenosine deaminases or evolved adenosine deaminases, described herein can be derived from any organism, including bacteria.
  • the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is TadA variant. In some embodiments, the TadA variant is a TadA8e. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism, such as bacteria, archaea, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not occur in nature.
  • the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase
  • TadA8e adenosine deaminase has the following sequence:
  • the amino acid substitution may be at least one selected from the group consisting of V28Q, or V28R, A48W, F84M, V106A, K110S, K110T, or K110V, and R111F, R111Q, R111S, R111T, or R111Y of the amino acid sequence of SEQ ID NO:1.
  • the amino acid substitution having the lowest RNA or DNA off-target editing efficiency may be at least one selected from the group consisting of V28Q, or V28R, A48W, and R111S, of the amino acid sequence of SEQ ID NO:1.
  • the adenine deaminase variant may exhibit remarkably reduced off-target effects involving an unintended base alteration in DNA and/or RNA.
  • the adenine deaminase variant may reduce unwanted bystander effects while narrowing activity windows.
  • the adenine deaminase variant may induce base editing only at a single nucleotide residue without any intended off-target editing in the target DNA with a frequency of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%.
  • a fusion protein comprising a DNA-binding protein; and the adenine deaminase variant.
  • the amino acid substitution of the adenine deaminase variant may be at least one selected from the group consisting of V28Q, or V28R, A48W, F84M, V106A, K110S, K110T, or K110V, and R111F, R111Q, R111S, R111T, or R111Y of the amino acid sequence of SEQ ID NO:1.
  • the amino acid substitution of the adenine deaminase variant having the lowest RNA or DNA off-target editing effects may be at least one selected from the group consisting of V28Q, or V28R, A48W, and R111S, of the amino acid sequence of SEQ ID NO:1.
  • the fusion protein may exhibits remarkably reduced off-target effects involving an unintended base alteration in DNA and/or RNA.
  • the fusion protein may reduce unwanted bystander effects while narrowing activity windows.
  • the fusion protein may induce base editing only at a single nucleotide residue without any intended off-target editing in the target DNA with a frequency of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%.
  • the DNA binding protein may be, for example, but is not limited to, 1) zinc finger protein, 2) transcriptional activator-like effector (TALE) protein, 3) CRISPR-associated nuclease.
  • the nuclease is a type II and / or type V such as Cas protein (e.g., Cas9 protein (CRISPR (Clustered regularly interspaced short palindromic repeats) associated protein 9)) or Cpf1 protein (CRISPR from Prevotella and Francisella 1).
  • Cas protein e.g., Cas9 protein (CRISPR (Clustered regularly interspaced short palindromic repeats) associated protein 9)
  • Cpf1 protein CRISPR from Prevotella and Francisella 1).
  • a nuclease associated with the CRISPR system for example, an endonuclease or the like may be used.
  • the nuclease may be a Cas protein such as Cas3, Cas9, Cpf1, Cas6, or C2c2, specifically the Cas protein of CRISPR/Cas type II, and more specifically a Cas9 protein derived from Streptococcus Pyogenes.
  • a Cas protein such as Cas3, Cas9, Cpf1, Cas6, or C2c2, specifically the Cas protein of CRISPR/Cas type II, and more specifically a Cas9 protein derived from Streptococcus Pyogenes.
  • TALE Transcriptional Activator-Like Effector
  • RVD Repeat Variable Diresidue
  • the RVD motif determines binding specificity to a nucleic acid sequence, and can be engineered according to methods well known to those of skill in the art to specifically bind a desired DNA sequence.
  • the simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
  • the DNA binding protein may be a TALE.
  • the TALE may be a dual TALE module consisting of a first TALE module and a second TALE module.
  • each of the first and second TALE modules may be connected to various deaminases.
  • the first TALE module may be linked to cytosine deaminase such as DddA tox in a full-length form
  • the second TALE module may be linked to the adenine deaminase variant.
  • Cas9 protein is a main protein component of the CRISPR/Cas system, which can function as an activated endonuclease or nickase.
  • Cas9 protein or gene information thereof may be acquired from a well-known database such as the GenBank of NCBI (National Center for Biotechnology Information).
  • the Cas9 protein may be at least one selected from the group consisting of, but not limited to:
  • Streptococcus pyogenes e.g., SwissProt Accession number Q99ZW2(NP_269215.1) (encoding gene: SEQ ID NO: 229);
  • a Cas9 protein derived from Streptococcus sp. for example, Streptococcus thermophiles or Streptocuccus aureus ;
  • Pasteurella multocida a Cas9 protein derived from Pasteurella sp., for example, Pasteurella multocida ;
  • a Cas9 protein derived from Francisella sp. for example, Francisella novicida .
  • Cpf1 protein which is an endonuclease of a new CRISPR system distinguished from the CRISPR/Cas system, is small in size compared to Cas9, requires no tracrRNA, and can function with a single guide RNA.
  • Cpf1 can recognize thymidine-rich PAM (protospacer-adjacent motif) sequences and produces cohesive double-strand breaks (cohesive end).
  • the Cpf1 protein may be an endonuclease derived from Candidatus spp., Lachnospira spp., Butyrivibrio spp., Peregrinibacteria , Acidominococcus spp., Porphyromonas spp., Prevotella spp., Francisella spp., Candidatus Methanoplasma ), or Eubacterium spp.
  • Examples of the microorganism from which the Cpf1 protien may be derived include, but are not limited to, Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus , Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae , Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis , Prevotella disiens , Moraxella bovoculi (237), Smiihella sp.
  • SC_KO8D17 Leptospira inadai , Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum , Candidatus Paceibacter, and Eubacterium eligens .
  • the Cas9 protein may be at least one selected from the group consisting of modified Cas9 that lacks endonuclease activity and retains nickase activity as a result of introducing mutations (e.g., substitution with a different amino acid) to D10 of Streptococcus pyogenes -derived Cas9 protein (e.g., SwissProt Accession number Q99ZW2(NP_269215.1)), and modified Cas9 protein that lacks both endonuclease activity and nickase activity as a result of introducing mutations (e.g., substitutions with different amino acids) to both D10 and H840 of Streptococcus pyogenes -derived Cas9 protein.
  • the mutation at D10 may be D10A mutation (the amino acid D at position 10 in Cas9 protein is substituted with A)
  • the mutation at H840 may be H840A
  • the nick may be introduced simultaneously with the diaminase-mediated base modification (e.g. cytidine converted to uradine) or sequentially, in any order, on the strand on which the base modification occurred or on the opposite strand thereof (e.g. strand opposite to the strand where base conversion occurred) (e.g., a nick is introduced at a position between the third nucleotide and the fourth nucleotide positions in the direction of the 5' end of the PAM sequence on the opposite strand of the strand where the PAM is located).
  • Nuclease mutations e.g., amino acid substitutions, etc.
  • can occur in the catalytically active domain of the nuclease e.g., in the case of Cas9, the RuvC catalytic domain).
  • the mutations may be a substitution of at least one amino acid selected from the group consisting of catalytic aspartic acid at position 10 (D10), glutamic acid at position 762 (E762), histidine at position 840 (H840), asparagine at position 854 (N854), asparagine at position 863 (N863), and aspartic acid at position 986 (D986) for another amino acid.
  • D10 catalytic aspartic acid at position 10
  • E762 glutamic acid at position 762
  • H840 histidine at position 840
  • N854 asparagine at position 854
  • N863 asparagine at position 863
  • D986 aspartic acid at position 986
  • it can include variants in which amino acids at N863, H840-N863, or H839-H840-N863 of Cas9 are replaced with another amino acid.
  • D10A SpCas9 nickase SpCas9 nickase prepared by removing some catalytic domains may also be used.
  • the fusion protein may further include a guide RNA.
  • the guide RNA may be, for example, at least one selected from the group consisting of CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and single guide RNA (sgRNA). Specifically, it may be a double-stranded crRNA:tracrRNA complex in which crRNA and tracrRNA are bonded to each other, or a single-stranded guide RNA (sgRNA) in which crRNA or a portion thereof and tracrRNA or a portion thereof are linked by an oligonucleotide linker.
  • crRNA CRISPR RNA
  • tracrRNA trans-activating crRNA
  • sgRNA single guide RNA
  • the adenine deaminase variant and the DNA binding protein may be used in the form of a fusion protein in which they are fused to each other directly or via a peptide linker (e.g., existing in the order of adenine deaminase variant-DNA binding protein in the N- to C-terminus direction (i.e. , DNA binding protein fused to the C-terminus of adenine deaminase variant) or in the order of DNA binding protein-adenine deaminase variant in the N- to C-terminus direction ( i.e.
  • a peptide linker e.g., existing in the order of adenine deaminase variant-DNA binding protein in the N- to C-terminus direction (i.e. , DNA binding protein fused to the C-terminus of adenine deaminase variant) or in the order of DNA binding protein-adenine deaminase variant in the N- to C-terminus
  • adenine deaminase variant fused to the C-terminus of DNA binding protein a mixture of the adenine deaminase variant or mRNA coding therefor and the DNA binding protein or mRNA coding therefor, a plasmid carrying both an adenine deaminase variant-encoding gene and a DNA binding protein-encoding gene (e.g., the two genes arranged to encode the fusion protein described above, or a mixture of a adenine deaminase variant expression plasmid and a DNA binding protein expression plasmid, or a plasmid which carry an adenine deaminase variant-encoding gene and an DNA binding protein-encoding gene, respectively).
  • a plasmid carrying both an adenine deaminase variant-encoding gene and a DNA binding protein-encoding gene e.g., the two genes arranged to encode the fusion protein described above, or a mixture of a adenine
  • the fusion protein may further comprise a cytosine deaminase.
  • the cytosine deaminase refers to any enzyme having activity to convert a cytosine, which is found in nucleotide (e.g., cytosine present in double stranded DNA or RNA), to uracil (C-to-U conversion activity or C-to-U editing activity).
  • the cytosine deaminase converts cytosine positioned on a strand where a PAM sequence linked to target sequence is present, to uracil.
  • the cytosine deaminase may be originated from mammals including bacteria, archaea, primates such as humans and monkeys, rodents such as rats and mice, and the like, but not be limited thereto.
  • the cytosine deaminase may be at least one selected from the group consisting of PmCDA1 (Petromyzon marinus cytosine deaminase 1) from Petromyzon marinus, DddA tox from Burkholderia cenocepacia , and APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family, but not be limited thereto.
  • the cytosine deaminase is wild-type Petromyzon marinus CDA1 (pmCDA1) or a catalytic domain thereof. In some embodiments, the cytosine deaminase comprises one or more mutations in the pmCDA1 sequence, such that the editing efficiency, and/or substrate editing preference of pmCDA1 is changed according to specific needs.
  • pmCDA1 has the following amino acid sequence:
  • DddA tox is cytotoxic, and thus, in order to avoid toxicity in host cells, DddA tox is split into two inactive halves, each of which is fused to a DNA-binding protein in a DddA-derived cytosine base editor (DdCBE).
  • DdCBE DddA-derived cytosine base editor
  • a functional deaminase is reassembled at a target DNA site, when two inactive halves are brought together by the DNA-binding protein.
  • the full-length DddA tox has the following amino acid sequence:
  • 6U08_A of Bu rkhol.de ria cenocepacia can include fragments or variants thereof, including amino acid sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identify with DddA of 6U08_A.
  • the APOBEC apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like family, for example, may be at least one selected from the following group, but not be limited to:
  • APOBEC1 Homo sapiens APOBEC1 (Protein: GenBank Accession Nos. NP_001291495.1, NP_001635.2, NP_005880.2, etc.; gene (mRNA or cDNA; described in the order of the above listed corresponding proteins): GenBank Accession Nos. NM_001304566.1, NM_001644.4, NM_005889.3, etc.), Mus musculus APOBEC1 (protein: GenBank Accession Nos. NP_001127863.1, NP_112436.1, etc.; gene: GenBank Accession Nos. NM_001134391.1, NM_031159.3, etc.);
  • APOBEC2 Homo sapiens APOBEC2 (protein: GenBank Accession No. NP_006780.1, etc.; gene: GenBank Accession No. NM_006789.3 etc.), mouse APOBEC2 (protein: GenBank Accession No. NP_033824.1, etc.; gene: GenBank Accession No. NM_009694. 3, etc.);
  • APOBEC3B Homo sapiens APOBEC3B (protein: GenBank Accession Nos. NP_001257340.1, NP_004891.4, etc.; gene: GenBank Accession Nos. NM_001270411.1, NM_004900.4, etc.), Mus musculus APOBEC3B (proteins: GenBank Accession Nos. NP_001153887.1, NP_001333970.1, NP_084531.1, etc.; gene: GenBank Accession Nos. NM_001160415.1, NM_001347041.1, NM_030255.3, etc.);
  • APOBEC3C Homo sapiens APOBEC3C (protein: GenBank Accession No. NP_055323.2 etc.; gene: GenBank Accession No. NM_014508.2 etc.);
  • APOBEC3D Homo sapiens APOBEC3D (protein: GenBank Accession No. NP_689639.2, etc.; gene: GenBank Accession No. NM_152426.3 etc.);
  • APOBEC3F Homo sapiens APOBEC3F (protein: GenBank Accession Nos. NP_660341.2, NP_001006667.1, etc.; gene: GenBank Accession Nos. NM_145298.5, NM_001006666.1, etc.);
  • APOBEC3G Homo sapiens APOBEC3G (protein: GenBank Accession Nos. NP_068594.1, NP_001336365.1, NP_001336366.1, NP_001336367.1, etc.; gene: GenBank Accession Nos. NM_021822.3, NM_001349436.1, NM_001349437.1, NM_001349438.1, etc.);
  • APOBEC3H Homo sapiens APOBEC3H (protein: GenBank Accession Nos. NP_001159474.2, NP_001159475.2, NP_001159476.2, NP_861438.3, etc.; gene: GenBank Accession Nos. NM_001166002.2, NM_001166003. 2, NM_001166004.2, NM_181773.4, etc.);
  • APOBEC4 (including APOBEC3E): Homo sapiens APOBEC4 (protein: GenBank Accession No. NP_982279.1, etc.; gene: GenBank Accession No. NM_203454.2 etc.); mouse APOBEC4 (protein: GenBank Accession No. NP_001074666.1, etc.; gene: GenBank Accession No. NM_001081197.1, etc.); and
  • Activation-induced cytidine deaminase Homo sapiens AID (Protein: GenBank Accession Nos. NP_001317272.1, NP_065712.1, etc; Genes: GenBank Accession Nos. NM_001330343 .1, NM_020661.3, etc.); mouse AID (protein: GenBank Accession No. NP_033775.1, etc., gene: GenBank Accession No. NM_009645.2, etc.), and the like.
  • the cytosine deaminase may be a non-toxic full-length deaminase (i.e. monomeric cytosine deaminase) or be in a two-split form ( i.e. dimeric deaminase) comprising separated first and second domains, each of which may be characterized by the absence of deaminase activity.
  • the adenine deaminase variant may bind to the N- or C-terminus of a DNA binding protein or cytosine deaminase or variant thereof.
  • the DNA-binding protein is ZFP
  • the adenine deaminase variant is TadA8e variant
  • the cytosine deaminase or its variant is DddA tox
  • they can be included in the following order, but are not limited thereto: ZFP-TadA8e variant- DddA tox , ZFP -DddA tox -TadA8e variant, TadA - DddA tox -ZFP, or DddAtox-TadA8e variant-ZFP.
  • the adenine deaminase variant may be attached to the C- terminus of the zinc finger protein (ZF-Left), the N-terminus or C-terminus of the first domain of the cytosine deaminase, the N-terminus of zinc finger protein (ZF-Right), or the N-terminus or C-terminus of the second domain of the cytosine deaminase.
  • the adenine deaminase variant may bind to:
  • the adenine deaminase variant may bind to C-terminus of zinc finger protein (ZF-Left), N-terminus or C-terminus of the first domain of the cytosine deaminase, zinc finger protein (ZF-Right), or N-terminal or C-terminal of the second domain of the cytosine deaminase.
  • the cytosine deaminase when the cytosine deaminase is in a split form and the DNA binding protein is a TALE, the first domain of the cytosine deaminase is attached to a first TALE, the second domain of the cytosine deaminase is attached to a second TALE, and each has a structure of N'-TALE-first domain DDDA-C' and N'-TALE-second domain DDDA-C', respectively.
  • the adenine deaminase variant may bind to the N-terminus or C-terminus of the first domain of cytosine deaminase or the N-terminus or C-terminus of the second domain of cytosine deaminase.
  • the cytosine deaminase when included in a full-length form and the DNA binding protein is a TALE, it can include a single TALE module, including a single TALE module and a cytosine deaminase in the NC orientation, wherein an adenine deaminase variant can bind to the C-terminus of the single TALE module, or to the N- or C-terminus of the cytosine deaminase.
  • cytosine deaminase when the cytosine deaminase is included in a full-length form and the DNA binding protein is a TALE, a dual TALE module may be included.
  • a first TALE module and cytosine deaminase are included in the N-C direction, and a second domain including an adenine deaminase variant and a second TALE may be further included.
  • the adenine deaminase variant can bind to the N-terminus or C-terminus of TALE.
  • the fusion protein may further comprise UGI (uracil glycosylase inhibitor).
  • UGI can increase the efficiency of base correction by inhibiting the activity of UDG (Uracil DNA glycosylase), an enzyme that repairs mutant DNA that catalyzes the removal of U from DNA.
  • the DNA may be nuclear DNA or organellar DNA.
  • the fusion protein may further comprise NLS (nuclear localization signal).
  • the nuclear localization signal protein may be, for example, derived from the simian virus 40 large tumor antigen (SV40 large T antigen), but is not limited thereto.
  • the nuclear localization signal protein may contain, for example, the following amino acid sequence, but is not limited thereto:
  • PKKKRKV (SEQ ID NO: 4)
  • the fusion protein may further comprise MTS (mitochondrial targeting sequence) or CTP (chloroplast transit peptide).
  • MTS mitochondrial targeting sequence
  • CTP chloroplast transit peptide
  • the mitochondrial targeting sequence protein may be, for example, SOD2-MTS or COX8A-MTS, and may contain the following amino acid sequences, but are not limited thereto:
  • SOD2-MTS LSRAVCGTSRQLAPVLGYLGSRQKHSLPD (SEQ ID NO: 5)
  • COX8A-MTS SVLTPLLLRGLTGSARRLPVPRAKIHSL (SEQ ID NO: 6).
  • the chloroplast transit peptide protein may be, for example, derived from Arabidopsis RECA1 but is not limited thereto.
  • the chloroplast transit peptide protein may contain, for example, the following amino acid sequence, but is not limited thereto:
  • the fusion protein may further comprise NES (nuclear export signal).
  • the nuclear export signal protein may be, for example, derived from MVM (Mirute virus of mice), but is not limited thereto.
  • the nuclear export signal protein may contain, for example, the following amino acid sequence, but is not limited thereto:
  • VDEMTKKFGTLTIHDTEK (SEQ ID NO: 8)
  • the structure when the signal peptides are attached to the fusion protein, the structure may be as follows: Signal peptide - DNA binding protein - Deaminase. In another embodiment, the structure could be Signal Peptide - Deaminase - DNA binding protein. In one embodiment, the nuclear export signal protein, CTP (chloroplast transit peptide), or a polynucleotide encoding the same may be attached to the N-terminus of a DNA-binding protein, cytosine deaminase (DdCBE), or a polynucleotide encoding the same.
  • CTP chloroplast transit peptide
  • DdCBE cytosine deaminase
  • the fusion protein may further comprise a nickase, for example, MutH, MutH variants, or Nt.BspD6I(C), but not limited thereto.
  • MutH is a weak endonuclease that is activated once bound to MutL. It nicks unmethylated DNA and the unmethylated strand of hemimethylated DNA but does not nick fully methylated DNA.
  • nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I.
  • the fusion protein further comprising a nickase may be in a dimeric form comprising a first fusion protein and a second protein.
  • the first fusion protein may include the DNA-binding protein, and the adenine deaminase variant, and the second protein may include another DNA-binding protein, and the nickase.
  • a polynucleotide encoding the adenine deaminase or the fusion protein is provided.
  • the term “polynucleotide” is used interchangeably with “nucleic acid”, “oligonucleotide”, “nucleotide”, “nucleotide sequence.” It can contain polymeric forms of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function.
  • a polynucleotide can comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure are possible before or after assembly of the polymer.
  • the nucleic acid can be an RNA sequence, in particular an mRNA sequence, a DNA sequence, or a combination thereof (RNA-DNA combination sequences).
  • the nucleic acid may be delivered using a viral vector such as Adeno-Associated Viral Vector (AAV), Adenoviral Vector (AdV), Lentiviral Vector (LV) Retroviral Vector (RV), or other viral vectors such as episomal vector including Simian virus 40 (SV40) ori, bovine papilloma virus (BPV) ori or Epstein-Barr nuclear antigen (EBV).
  • AAV Adeno-Associated Viral Vector
  • AdV Adenoviral Vector
  • LV Lentiviral Vector
  • RV Retroviral Vector
  • the delivery may be also carried out using a non-viral vector, or through plasmid or mRNA delivery.
  • the vector may be delivered in vivo or into cells by a local injection method (e.g., direct injection into a lesion or target site), electroporation, lipofection, viral vector, nanoparticles, PTD (protein translocation domain) fusion protein method, or the like.
  • a local injection method e.g., direct injection into a lesion or target site
  • electroporation e.g., electroporation, lipofection, viral vector, nanoparticles, PTD (protein translocation domain) fusion protein method, or the like.
  • PTD protein translocation domain
  • known expression vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors can be used.
  • vectors can be readily prepared by those skilled in the art according to any known method using DNA recombination techniques.
  • a recombinant expression vector is designed to carry nucleic acid in a format that facilitates its expression within a host cell.
  • the nucleic acid sequence intended for expression is operably linked to the recombinant expression vector, and this vector is equipped with one or more regulatory elements, which can be chosen according to the specific host cell.
  • "operably linked" means that the nucleotide sequence of interest is linked to a regulatory element in a manner that allows expression of the nucleotide sequence. (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced to the host cell).
  • a base editing composition for A-to-G base editing in DNA comprising the fusion protein, a polynucleotide encoding the fusion protein or an expression vector comprising the polynucleotide.
  • the fusion protein has been explained above in detail.
  • the fusion protein of the base editing composition may further comprise a cytosine deaminase.
  • the cytosine deaminase has been explained above.
  • the cytosine deaminase may be present in a two split form, and the fusion protein may comprise a first fusion protein comprising a first split of the cytosine deaminase and a second fusion protein comprising a second split of the cytosine deaminase.
  • the first split may comprise the amino acid sequence of SEQ ID NO: 9 or 10
  • the second split may comprise the amino acid sequence of SEQ ID NO: 11 or 12, but is not limited thereto.
  • two TALEDs may consist of the left- or right-side TALE fused to the N-terminal DddAtox half split at G1397 (L-1397N or R-1397N, respectively) and the right- or left-side TALE fused to the C-terminal DddAtox half split at G1397 and TadA8e (R-1397C-AD or L-1397C-AD).
  • the base editing composition may exhibits remarkably reduced off-target effects involving an unintended base alteration in DNA and/or RNA.
  • the base editing composition may reduce unwanted bystander effects while narrowing activity windows.
  • the base editing composition may induce base editing only at a single nucleotide residue without any intended off-target editing in the target DNA with a frequency of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%.
  • a method for A-to-G base editing in DNA comprising delivering the base editing composition to a cell containing a target DNA.
  • the cell may be eukaryotic cells (e.g., fungi such as yeast, eukaryotic animals and/or eukaryotic plant-derived cells (e.g., embryonic cells, stem cells, somatic cells, gametes, etc.), eukaryotic animals (e.g., humans, monkeys, primates dogs, pigs, cattle, sheep, goats, mice, rats, etc.), or eukaryotic plants (e.g., algae such as green algae, corn, soybeans, wheat, rice, etc.), but is not limited thereto.
  • fungi such as yeast
  • eukaryotic animals and/or eukaryotic plant-derived cells e.g., embryonic cells, stem cells, somatic cells, gametes, etc.
  • eukaryotic animals e.g., humans, monkeys, primates dogs, pigs, cattle, sheep, goats, mice, rats, etc.
  • eukaryotic plants e.g., algae such as green algae, corn, soybean
  • the delivery of the base editing composition to a cell containing a target DNA may be carried out ex vivo or in vivo.
  • the target DNA may be nuclear DNA, organellar DNA, or mitochondrial DNA of a human subject with a hereditary disease.
  • hereditary disease refers to a pathological condition that occurs due to a mutation that is harmful to a gene or chromosome.
  • hereditary diseases include, but are not limited to, mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, DEAF, leber hereditary optic neuropathy (LHON), leigh Syndrome, Myopath and Chronic progressive external ophthalmoplegia (CPEO)
  • the method may method may exhibit reduced off-target effects compared to a case when using a base editor comprising an adenine deaminase having the amino acid sequence of SEQ ID NO:1, wherein the off-target editing is characterized by an unintended base alteration in DNA and/or RNA.
  • the method may reduce unwanted bystander effects while narrowing activity windows compared to when using a base editor comprising an adenine deaminase having amino acids represented by SEQ ID:1 mutation.
  • the method may induce base editing only at a single nucleotide residue without any intended off-target editing in the target DNA with a frequency of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%.
  • the method may exhibit a reduced off-target effect compared to when using a base editor comprising an adenine deaminase having amino acids represented by SEQ ID:1 with V106W mutation, wherein the off-target editing is characterized by an unintended base alteration in DNA and/or RNA.
  • the method may reduce unwanted bystander effects while narrowing activity windows compared to when using a base editor comprising an adenine deaminase having amino acids represented by SEQ ID:1 with V106W mutation.
  • the method may induce base editing only at a single nucleotide residue without any intended off-target editing in the target DNA with a frequency of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%.
  • a method for reducing off-target effect and/or unwanted bystander effects while narrowing activity windows comprising delivering the base editing composition to a cell containing a target DNA.
  • the cell may be eukaryotic cells (e.g., fungi such as yeast, eukaryotic animals and/or eukaryotic plant-derived cells (e.g., embryonic cells, stem cells, somatic cells, gametes, etc.), eukaryotic animals (e.g., humans, monkeys, primates dogs, pigs, cattle, sheep, goats, mice, rats, etc.), or eukaryotic plants (e.g., algae such as green algae, corn, soybeans, wheat, rice, etc.), but is not limited thereto.
  • fungi such as yeast
  • eukaryotic animals and/or eukaryotic plant-derived cells e.g., embryonic cells, stem cells, somatic cells, gametes, etc.
  • eukaryotic animals e.g., humans, monkeys, primates dogs, pigs, cattle, sheep, goats, mice, rats, etc.
  • eukaryotic plants e.g., algae such as green algae, corn, soybean
  • the target DNA may be nuclear DNA, organellar DNA, or mitochondrial DNA of a human subject with a hereditary disease.
  • the hereditary disease has been defined above.
  • the method may method may exhibit reduced off-target effects compared to a case when using a base editor comprising an adenine deaminase having the amino acid sequence of SEQ ID NO:1, wherein the off-target editing is characterized by an unintended base alteration in DNA and/or RNA.
  • the method may reduce unwanted bystander effects while narrowing activity windows compared to when using a base editor comprising an adenine deaminase having amino acids represented by SEQ ID:1.
  • the method may induce base editing only at a single nucleotide residue without any intended off-target editing in the target DNA with a frequency of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%.
  • the method may exhibit a reduced off-target effect compared to when using a base editor comprising an adenine deaminase having amino acids represented by SEQ ID:1 with V106W mutation, wherein the off-target editing is characterized by an unintended base alteration in DNA and/or RNA.
  • the method may reduce unwanted bystander effects while narrowing activity windows compared to when using a base editor comprising an adenine deaminase having amino acids represented by SEQ ID:1 with V106W mutation.
  • the method may induce base editing only at a single nucleotide residue without any unintended off-target editing in the target DNA with a frequency of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%.
  • adenine deaminase variant or the fusion protein in A-to-G base editing in DNA and/or reducing off-target effect in A-to-G base editing in DNA, or in preparing a composition for A-to-G base editing in DNA and/or reducing off-target effect in A-to-G base editing in DNA.
  • the adenine deaminase variant and the fusion protein have been explained above in detail.
  • the fusion protein may further comprise a cytosine deaminase.
  • the cytosine deaminase has been explained above.
  • the cytosine deaminase may be present in two split form, and the fusion protein comprises a first fusion protein comprising a first split of the cytosine deaminase and a second fusion protein comprising a second split of the cytosine deaminase.
  • the first split may comprise the amino acid sequence of SEQ ID NO: 9 or 10 and the second split may comprise the amino acid sequence of SEQ ID NO: 11 or 12, but is not limited thereto.
  • the composition for A-to-G base editing in DNA and/or reducing off-target effect in A-to-G base editing in DNA may exhibit remarkably reduced off-target effects involving an unintended base alteration in DNA and/or RNA.
  • the composition may reduce unwanted bystander effects while narrowing activity windows Alternatively, the composition may induce base editing only at a single nucleotide residue without any unintended off-target editing in the target DNA with a frequency of at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, or at least 15%.
  • HEK 293T cells were purchased from the American Type Culture Collection (ATCC) (CRL-11268). NIH3T3 and B16F10 cells were purchased from ATCC (CRL-1658, CRL-6475). HEK 293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; Welgene) supplemented with 10% (v/v) fetal bovine serum (Welgene) and 1% (v/v) antibiotic-antimycotic solution (Welgene).
  • DMEM Dulbecco's Modified Eagle Medium
  • NIH3T3 and B16F10 cells were cultured in DMEM supplemented with 10% (v/v) bovine calf serum (Gibco) for NIH3T3 cells or 10% fetal bovine serum (Gibco) for B16F10 cells in the absence of any antibiotics. The cells were incubated at 37°C with 5% CO 2 . All cell lines were passaged before approaching 90% confluency.
  • the ABE8e (PDB accession number 6VPC) structure was downloaded from PDB and visualized with PyMOL v.2.5.4. Some elements including Cas9, single guide RNA, and double-stranded DNA were excluded from the PDB file; 8AZ, a transition state analog for adenosine deamination reactions, and the TadA monomer were retained. 11 residues (V28, V30, N46, A48, I49, V82, F84, V106, N108, K110, and R111) that were near 8AZ, including residues previously known to contact DNA, were selected.
  • plasmids encoding DdCBEs, DddA-split TALEDs (sTALEDs), and mTALEDs that target specific sites plasmids containing a stuffer, which is a sequence between two restriction enzyme sites that is helpful when separating fragments during gel electrophoresis were prepared.
  • the architectures of the plasmids are as follows: sTALED plasmids (p3s-stuffer-DddA tox half (1397C)-AD and p3s-stuffer-DddA tox half (1397N)); mTALED plasmids (p3s-stuffer-E1347A DddA tox full-AD).
  • Plasmids encoding DdCBEs, sTALEDs, and mTALEDs targeting specific sites were constructed by inserting custom-designed TALE array sequences as shown in Table 1 below.
  • each plasmid digested with BsaI or BsmbI (NEB) and inserts, including a custom-designed TALE array sequence synthesized by IDT, were inserted into the digested vector using a HiFi DNA assembly kit (NEB).
  • the desired TALED construct was generated by digesting the master vector with BsaI or BsmbI to cleave the site in the stuffer and then assembling six TALE arrays in that position using the Golden Gate method.
  • HEK 293T cells were grown in DMEM (Welgene) with 10% fetal bovine serum (Welgene) and 1% antibiotic-antimycotic solution (Welgene).
  • NIH3T3 (CRL-1658, ATCC) and B16F10 (CRL-6475, ATCC) cells were grown in DMEM supplemented with 10% (v/v) bovine calf serum (Gibco) for NIH3T3 cells or 10% fetal bovine serum (Gibco) for B16F10 cells in the absence of any antibiotics.
  • Cell lines were maintained at 37°C with 5% CO 2 and were passaged before reaching 90% confluency, with timing depending on the doubling period of the specific cell line.
  • HEK 293T cells For HEK 293T cells, cells were seeded onto 48-well plates (Corning) at a density of 7.5 ⁇ 10 4 cells per well prior to transfection. The cells were transfected with plasmids (total 1ug) using 1.5 uL of Lipofectamine 2000 (Invitrogen) after 24 h. For delivery of sTALED pair, DdCBE pair, and CRISPR-Base Editor with sgRNA, the total amount of plasmid was 1 ug (500 ng each). When single construct was used, the amount of transfected plasmid was 500 ng. After 96 h, the transfected cells were harvested.
  • NIH3T3 and B16F10 cells were seeded in 24-well cell culture plates (SPL, Seoul, Korea) at a density of 1 ⁇ 10 5 cells per well, 18-24 h before transfection.
  • Lipofection using Lipofectamine 3000 was performed with 500 ng of each sTALED-encoding plasmid to make up 1000 ng of total plasmid DNA.
  • mTALED 500 ng of plasmid was used.
  • Cells were harvested 3 days after transfection.
  • RNA libraries were prepared using a TruSeq Stranded Total RNA Library Prep Gold kit (Illumina). RNA library quality was assessed using a 2200 TapeStation with a D1000 ScreenTape system (Agilent).
  • Total RNA sequencing was performed using a NovaSeq 6000 Sequencer (Illumina) at Macrogen with paired-end sequencing systems (2 x 100bp).
  • RNA base-editing variants were called using GATK HaplotypeCaller.
  • RNA variant loci were compared to those of control samples and filtered based on the following criteria: (1) loci with a read depth of at least 10 were retained; (2) loci with a variant count of at least 2 were retained; (3) loci also present in the control sample were removed; and (4) undeterminable loci due to insufficient sequencing depth in the control sample were excluded.
  • untreated replicate-2 was used as the control for filtering, and for the replicate-2 experimental sets, untreated replicate-1 was used as the control.
  • A-to-G edits were counted the number of RNA variant loci with A-to-G edits on the positive strand or T-to-C edits on the negative strand.
  • C-to-T edits were counted the number of RNA variant loci with C-to-T edits on the positive strand or G-to-A edits on the negative strand.
  • HEK 293T cells were treated with 100 ⁇ L of cell lysis buffer (50mM Tris-HCl; pH 8.0, 1mM EDTA, 0.005% sodium dodecyl sulfate) supplemented with 5 ⁇ L of Proteinase K (Qiagen). The cells were lysed by incubation at 55°C for 1h, and then at 95°C for 10min. The genomic DNA mixture was subjected to targeted deep sequencing.
  • cell lysis buffer 50mM Tris-HCl; pH 8.0, 1mM EDTA, 0.005% sodium dodecyl sulfate
  • NGS Libraries for targeted deep sequencing were created using nested PCR.
  • the target area was initially amplified by PCR using PrimeSTAR® GXL polymerase (Takara). Amplicons were amplified again by PCR with TruSeq DNA-RNA CD index-containing primers to label each fragment with adapter and index sequences in order to build NGS libraries.
  • the PCR primers are listed in Table 2 below.
  • the final PCR products were purified with a PCR purification kit (MGmed) and sequenced using a MiniSeq sequencer (Illumina). Base editing frequencies from targeted deep sequencing data were measured with source code (https://github.com/ibs-cge/maund).
  • the DNA on-target activity and RNA off-target activity of the variants were normalized to the sTALED values. Then, the normalized DNA on-target value is divided by the normalized RNA off-target value, as shown below.
  • this value is 1 because both the normalized DNA on-target value and RNA off-target value are 1. The higher the relative ratio indicates lower the off-target activity compared to the on-target activity.
  • PCR amplification For whole mitochondrial genome sequencing, three procedures were required: PCR amplification, NGS library creation, and NGS. Initially, cells were treated with 100 ⁇ L of cell lysis buffer (50mM Tris-HCl; pH 8.0, 1mM EDTA, 0.005% sodium dodecyl sulfate) supplemented with 5 ⁇ L of Proteinase K (Qiagen) after removing the growth medium. The cells were lysed by incubation at 55°C for 1h, and then at 95°C for 10min. Mitochondrial DNA was then amplified by PCR using PrimeSTAR® GXL polymerase (Takara). PCR was performed using two sets of slightly overlapping primers in shown in Table 2 above to reduce primer bias.
  • cell lysis buffer 50mM Tris-HCl; pH 8.0, 1mM EDTA, 0.005% sodium dodecyl sulfate
  • Qiagen Proteinase K
  • Mitochondrial DNA was then ampl
  • PCR products were then purified with a PCR purification kit (MGmed). Finally, an Illumina DNA Prep kit with Nextera DNA CD Indexes was used to create an NGS library from the purified PCR products (Illumina). The libraries were then pooled and transferred onto a MiniSeq sequencer (Illumina).
  • the remaining sites were regarded as off-target sites, and we counted the number of edited A/T nucleotides with an editing frequency > 0.1%.
  • the average A/T to G/C editing frequency were calculated for all bases in the mitochondrial genome by averaging the conversion rates at each base location in the off-target sites, as shown below.
  • Mitochondrial genome-wide graphs were constructed by plotting the conversion rates at on-target and off-target sites with an editing frequency ⁇ 1% across the entire mitochondrial genome.
  • Cell viability assays were performed using CellTiter 96® Aqueous One Solution (Promega) at day 2 or day 4 after plasmid transfection.
  • the MTS assay measured the number of viable cells with a colorimetric method.
  • Cells were treated with CellTiter 96® Aqueous One Solution, and the quantification of bio-reduced product was measured by recording the absorbance at 490 nm according to the manufacturer’s instructions.
  • transcriptome-wide off-target A-to-I conversions induced by TALEDs site-specific mutations were introduced in TadA8e, including V106W, V106G, K20A/R21A (dual mutations), or F148A, which are known to reduce off-target RNA editing when incorporated in CRISPR RNA-guided adenine base editors (ABEs).
  • ABEs CRISPR RNA-guided adenine base editors
  • Transcriptome-wide sequencing showed that sTALED variants incorporating these mutations in TadA8e reduced the number of off-target A-to-G edits significantly but not completely, while retaining DNA on-target editing efficiencies (FIG. 1).
  • TALEDs were engineered by mutating amino-acid residues, including V106, at the substrate binding site in TadA8e.
  • plasmids encoding each of the resulting sTALED pairs were transfected into HEK 293T cells.
  • RNA off-target editing frequencies measured by targeted deep sequencing were in good agreement with those estimated by transcriptome-wide sequencing (FIGs 4(b) and 4(c)).
  • a total of 12 TadA8e variants were chosen, which minimized RNA off-target editing efficiencies at the six representative sites, while retaining mtDNA on-target editing efficiencies (FIG. 2(b)).
  • Example 3 Engineered TALEDs reduce bystander and off-target editing
  • sTALED variants with site-specific mutations at the TadA8e substrate-binding site could also reduce bystander editing at the target site and off-target editing in the mitochondrial genome, because these mutations could potentially fine-tune adenine deaminase activity for DNA substrates in addition to reducing activity for RNA substrates. That said, base editing frequencies at each nucleotide position were examined. Both sTALED-V28R and -R111S variants specific to the Cox3.1, ND1, and ND6 sites induced A-to-I edits in a narrower window than did the wild-type sTALEDs and sTALED-V106W variants (FIG. 7).
  • the wild-type sTALED and sTALED-v106W targeted to the Cox3.1 site induced A-to-I edits at multiple positions, with frequencies of >1.1%, not only in the spacer region between the two TALE-binding sites but also in the TALE-binding sites.
  • the Cox3.1- specific sTALED-V28R induced an A-to-I edit at a single position in the spacer region (FIG. 7a).
  • TALEDs inducing single-base substitutions with no or few bystander edits are desired, because the vast majority of pathogenic mitochondrial DNA mutations, responsible for mitochondrial genetic disorders, are single-nucleotide variations rather than multiple-nucleotide variations.
  • the number of off-target edits induced in the mitochondrial genome was also reduced by the sTALED variants.
  • the ND1 -specific, wild-type sTALED caused A-to-I off-target edits at 108 sites in human mitochodrial DNA with frequencies of > 0.1%
  • sTALED-V28R and -R111S induced off-target edits at 14 and 17 sites, respectively, similar to the baseline number (that is, 24) seen in the untreated sample (FIG. 8(a)).
  • Whole mitochondrial genome sequencing at day 2 post-transfection was also performed (FIG. 9), when RNA off-target edits were induced at the highest level.
  • FIG. 10 it was investigated whether on-target mutations induced by various forms of sTALEDs were stably maintained and how long RNA off-target variations persisted over time.
  • RNA edits were heavily induced by sTALEDs and sTALED-V106W variants at day 1 and 2 post-transfection but almost completely disappeared by day 8 post-transfection.
  • the new sTALED variants reduced RNA off-target editing frequencies by several fold, compared to sTALEDs or the sTALED-V106W variants, even at day 1 and 2 post-transfection (FIG. 10(g)). Mitochondria DNA on-target edits induced by the sTALEDs and sTALED-V106W variants were not stably maintained. Thus, the frequencies of mitochondria DNA on-target edits induced by the sTALEDs and sTALED-V106W variants dropped significantly over time.
  • mitochondria DNA on-target edits were induced by the ND6 -specific sTALED and sTALED-V106W with high frequencies of up to 47% and 40%, respectively, at day 1 and 2 post-transfection but with low frequencies of ⁇ 10% at day 8 or later (FIG. 10(c)).
  • the frequencies of on-target edits induced by sTALED-V28R and -R111S increased or were more stably maintained over time.
  • the editing frequencies observed for our new sTALED variants were lower at day 1 and 2 post-transfection but were higher at day 8 or later than those induced by previous versions of sTALEDs (FIG. 10(a)-(c)).
  • sTALEDs and sTALED-V106W variants were cytotoxic, because they induced too many RNA off-target edits with high frequencies, and that mtDNA-edited cells could not divide or died out over time.
  • sTALED variants of the present invention could be tolerated, possibly because they avoided RNA off-target editing or did not induce too many bystander edits at the target site and off-target mutations in the mitochondrial genome.
  • MTS cell proliferation assays FIG. 11(a) were performed to confirm that the sTALEDs and sTALED-V106W variants were indeed cytotoxic, reducing cell viability significantly, compared to the negative control (pEGFP transfection) (FIG. 11(b)). The sTALED variants were tolerated much better, such that cell viability was not reduced at day 4 post-transfection (FIG. 11(c)).
  • FIG. 12 it was investigated whether the V28R and R111S mutations in TadA8e could also reduce bystander editing and RNA off-target editing induced by CRISPR RNA-guided ABEs, widely used for nuclear DNA editing.
  • on-target and bystander editing frequencies at the TYRO3 site were measured.
  • Both ABE8e-V28R and -R111S were as efficient as ABE8e and ABE8e-V106W (ABE8eW) at the target site with editing frequencies of > 30% (FIGs. 12(b) and 12(c)).
  • the V28R and R111S variants exhibited a narrowed editing window with efficient editing (up to 37%) at positions 5 and 7 (A5 and A7 in FIG.
  • ABE8e and ABE8eW showed a broader editing window with maximum editing at A5 (29% and 30%, respectively) and A7 (29% and 30%, respectively) and substantial bystander editing at A10 (9.3% and 3.1%, respectively).
  • RNA off-target editing activities were used to assess RNA off-target editing activities.
  • ABE8e-V28R and -R111S reduced average RNA off-target editing frequencies measured at a total of 6 sites by 3.8 fold and 2.5 fold, respectively, compared to ABE8e, and 3.1 fold and 2.0 fold, compared to ABE8eW (FIG. 12(d)).
  • whole transcriptome sequencing showed that ABE8e-V28R and -R111S reduced the number of RNA off-target edits substantially, compared to ABE8e and ABE8eW (FIGs. 12(e) and 12(f)).
  • Example 6 DNA on-target and RNA off-target editing by mTALEDs and sTALEDs
  • each TALE unit contains a cytosine deaminase (e.g. DddA tox variant (E1347A)) on one side and an adenine deaminase (e.g. TadA8e) on the other side.
  • cytosine deaminase e.g. DddA tox variant (E1347A)
  • adenine deaminase e.g. TadA8e
  • FIG. 13(a) it was confirmed that both dTALEDs and mTALEDs targeted to Cox3 site exhibited on-target activity. Furthermore, it was observed that gene editing did not occur when only the adenine deaminase was present in the TALE unit.
  • FIG. 13(b) relates to the results of editing efficiency at six representative sites. Similar to the sTALEDs, a significant reduction in RNA off-target efficiencies for both dTALEDs and mTALEDs variants with V28R and R111S was observed compared to the TadA8e.
  • RNA off-target editing was analyzed.
  • the results demonstrate that the TadA variants with V28R and R111S, not only reduced the RNA off-target efficiency in sTALED and other systems like ABE but also in dTALEDs and mTALEDs as well.
  • RNA off-target effects were predominantly induced by the TadA adenine deaminase rather than by a DNA binding proteins or cytosine deaminase.
  • the TadA variants can remarkably reduce such undesired RNA off-target effects.

Abstract

La présente invention concerne un nouveau variant d'adénine désaminase, une protéine de fusion comprenant le variant d'adénine désaminase, une composition d'édition de bases pour l'édition de bases A en G dans l'ADN comprenant la protéine de fusion, et un procédé d'édition de bases A en G dans l'ADN comprenant l'administration de la composition d'édition de bases à une cellule contenant un ADN cible. Le nouveau variant d'adénine désaminase peut entraîner une réduction remarquable des effets hors cible impliquant une altération involontaire des bases dans l'ADN et/ou l'ARN, et induire une édition de bases uniquement au niveau d'un seul résidu nucléotidique sans aucune édition hors cible involontaire dans l'ADN cible.
PCT/KR2023/009361 2022-09-23 2023-07-04 Nouveaux variants d'adénine désaminase et procédé d'édition de bases les utilisant WO2024063273A1 (fr)

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US20200308571A1 (en) * 2019-02-04 2020-10-01 The General Hospital Corporation Adenine dna base editor variants with reduced off-target rna editing
WO2021050571A1 (fr) * 2019-09-09 2021-03-18 Beam Therapeutics Inc. Nouveaux éditeurs de nucléobases et leurs procédés d'utilisation
CN114045277A (zh) * 2021-10-21 2022-02-15 复旦大学 碱基编辑器及其构建方法与应用
WO2022119294A1 (fr) * 2020-12-01 2022-06-09 한양대학교 산학협력단 Éditeur de base d'adénine dépourvu d'activité d'édition de cytosine et son utilisation

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US20200308571A1 (en) * 2019-02-04 2020-10-01 The General Hospital Corporation Adenine dna base editor variants with reduced off-target rna editing
WO2021050571A1 (fr) * 2019-09-09 2021-03-18 Beam Therapeutics Inc. Nouveaux éditeurs de nucléobases et leurs procédés d'utilisation
WO2022119294A1 (fr) * 2020-12-01 2022-06-09 한양대학교 산학협력단 Éditeur de base d'adénine dépourvu d'activité d'édition de cytosine et son utilisation
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