Engineered adenosine deaminases and base editors

Abstract

Disclosed are engineered adenosine deaminases, base editors based on said adenosine deaminases, and complexes comprising said base editors. The described adenosine deaminases and base editors exhibit good adenine editing effects, and realize the DNA-level conversion of A•T base pairs to G•C base pairs.

Description

Engineered adenosine deaminase and base editors Technical Field

[0001] The present disclosure relates to engineered adenosine deaminases, base editors based on these adenosine deaminases, and complexes comprising these base editors. The present disclosure also relates to polynucleotides encoding engineered adenosine deaminases, codon-optimized polynucleotides, vectors comprising these nucleotides, and cells comprising these vectors. The present disclosure also relates to pharmaceutical compositions comprising the base editors, complexes, vectors, or cells, and methods and uses of treating diseases using the base editors, complexes, vectors, cells, or pharmaceutical compositions. Background Art

[0002] In 2016, scientists first developed base editing (BE) technology based on the CRISPR-Cas9 system, combining chemical biology with gene editing methods. This technology can precisely convert single bases without generating double-strand breaks (DsBs), effectively circumventing the shortcomings of gene editing technologies caused by non-homologous end joining (NHEJ) and homology directed repair (HDR) after double-strand breaks. Subsequently, scientists developed a new base editor, adenine base editor (ABE), based on E. coli TadA (ecTadA), which can precisely convert A to G at the target site and T to C on the complementary strand. At present, the ABE system is widely used in basic biology and medicine, mainly in the following aspects: (1) achieving precise editing of non-dividing cells; (2) screening of gene function; (3) creation and correction of premature stop codons; (4) identification of conserved functional amino acids; (5) creation of splice site variants; (6) accelerating the study of loss-of-function or gain-of-function at single base sites; and (7) clinical treatment and basic theoretical research of genetic diseases caused by single base mutations.

[0003] Current adenine base editors (ABEs) are composed of an RNA-guided Cas protein fused to an adenosine deaminase that acts on single-stranded DNA (ssDNA). When the fusion protein targets genomic DNA under the guidance of a guide RNA (such as sgRNA), the adenosine deaminase can bind to the ssDNA and deaminate adenine (A) at a specific position to inosine (I). I is read and replicated as G at the DNA level, ultimately achieving a direct replacement of the A·T base pair with the G·C base pair. The current ABE7.10 and miniABEmax require a long time to complete the reaction, are very inefficient, and run slowly. Scientists have further improved ABE7.10 and obtained ABE8e, which contains a single deaminase structure (TadA-8e) and can deaminate multiple adenines simultaneously. However, there are currently few types of adenosine deaminases and base editors that can efficiently deaminate multiple adenines, limiting their commercial application.

[0004] Summary of the Invention

[0005] One aspect of the present invention provides an adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 1: W23, Y36, P48, H51, L84, A106, D108, V109, K110, T111, D119, G122, H123, S146, F149, R152, H156, K157, E168 and E169, and having an amino acid sequence with about 70% to about 99.5% sequence identity to the sequence shown in SEQ ID NO: 1.

[0006] One aspect of the present invention provides an adenosine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 2 to 10, or an amino acid sequence having at least about 80% sequence identity thereto.

[0007] One aspect of the present invention provides an adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 12: E22, T47, L83, A105, D107, F108, T110, D118, R121, S145, F148, R151, E154, K156, V167 and E168, and having an amino acid sequence with about 70% to about 99.5% sequence identity to the sequence shown in SEQ ID NO: 12.

[0008] One aspect of the present invention provides an adenosine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 13 to 16, or an amino acid sequence having at least about 80% sequence identity thereto.

[0009] One aspect of the present invention provides an adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 17: W22, Q35, P47, Y50, L83, A105, D107, E108, T110, D118, G121, H122, S145, F148, R151, E154, K155 and K156, and having an amino acid sequence with about 70% to about 99.5% sequence identity to the sequence shown in SEQ ID NO: 17.

[0010] One aspect of the present invention provides an adenosine deaminase comprising an amino acid sequence selected from any one of SEQ ID NOs: 18 to 21 or an amino acid sequence having at least about 80% sequence identity thereto.

[0011] One aspect of the present invention provides a base editor, which comprises any one of the adenosine deaminase provided by the present invention; and a complex formed by the base editor and a guide RNA.

[0012] Other aspects of the present invention provide polynucleotides encoding any one of the adenosine deaminase or base editor provided by the present invention; vectors comprising the polynucleotides; cells comprising the vectors; and pharmaceutical compositions comprising the base editors, the complexes, the vectors or the cells.

[0013] The present invention also provides a method for treating a disease using a polynucleotide of any one of the adenosine deaminase or base editor provided by the present invention; a vector comprising the polynucleotide; a cell comprising the vector; or a pharmaceutical composition comprising the base editor, the complex, the vector or the cell.

[0014] The present invention also provides a polynucleotide of any one of the adenosine deaminase or base editor provided by the present invention; a vector comprising the polynucleotide; a cell comprising the vector; or a pharmaceutical composition comprising the base editor, the complex, the vector or the cell in the preparation of a drug for treating a disease.

[0015] The present invention also provides a polynucleotide of any one of the adenosine deaminase or base editor provided by the present invention for treating a disease; a vector comprising the polynucleotide; a cell comprising the vector; or a pharmaceutical composition comprising the base editor, the complex, the vector or the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1. The editing effect of some adenine base editors of the present invention on the start codon of the ccdB gene is shown by observing whether Escherichia coli can grow on solid culture medium.

[0017] Figure 2. The editing effect of some adenine base editors of the present invention on the termination codon of the ccdB gene is shown by observing whether Escherichia coli can grow on solid culture medium.

[0018] Figure 3. Sequencing shows the editing effects of some adenine base editors of the present invention on the start codon of the ccdB gene.

[0019] Figure 4. Sequencing shows the editing effects of some adenine base editors of the present invention on the stop codon of the ccdB gene.

[0020] Figure 5. Sequencing shows the editing effects of other adenine base editors of the present invention on the start codon of the ccdB gene.

[0021] Figures 6 to 10 show sequencing images of various adenine base editors editing the HEK2 gene in 293T cells.

[0022] Figures 11 to 15 show sequencing images of various adenine base editors editing the p992L gene in 293T cells.

[0023] Figures 16 to 22 show schematic structural diagrams of various base editor plasmids constructed according to embodiments of the present invention. DETAILED DESCRIPTION

[0024] definition

[0025] The term "base editor (BE)" as used herein refers to a reagent comprising a polypeptide capable of modifying a base (such as A, T, C, G or U) in a nucleic acid sequence (such as DNA or RNA). In some embodiments, the base editor is capable of deaminating bases in nucleic acids (such as DNA molecules). In the case of an adenine base editor (or adenosine editor), the base editor is capable of deaminating adenine (A) in DNA. Such a base editor may include a programmable DNA binding protein fused to an adenosine deaminase. Programmable DNA binding proteins include CRISPR-mediated Cas effector proteins. In some embodiments, the base editor includes a nuclease-inactive Cas9 (dCas9) fused to a deaminase, which binds to nucleic acids but does not cut nucleic acids. For example, the dCas9 domain of a base editor may include D10A and H840A mutations. The DNA cleavage domain of Bacillus Cas9 includes two subdomains, the HNH nuclease subdomain and the RuvC I subdomain. The HNH subdomain cuts the strand complementary to the gRNA (the "targeting strand," or the strand that undergoes editing or deamination), while the RuvC I subdomain cuts the non-complementary strand containing the PAM sequence (the "non-editing strand"). The RuvC I mutant D10A creates a nick in the targeting strand, while the HNH mutant H840A creates a nick on the unedited strand. In the present invention, the term "base editor" refers to a base editor comprising an adenosine deaminase domain, such as an adenine base editor (ABE) or a cytosine and adenine base editor (CABE).

[0026] The term "Cas9" or "Cas9 nuclease" or "Cas9 domain" refers to CRISPR-associated protein 9 or a variant thereof, including any native Cas9 from any organism, any native Cas9 equivalent or fragment thereof, any Cas9 homolog, ortholog or paralog from any organism, and any naturally occurring or engineered Cas9 variant. The term Cas9 is not limited to any specific Cas9 and may be referred to as "Cas9 or a variant thereof." Exemplary Cas9 proteins are described herein.

[0027] As used herein, the term "dCas9" refers to nuclease-inactive Cas9 or nuclease-dead Cas9, or variants thereof, and includes any naturally occurring dCas9 from any organism, any naturally occurring dCas9 equivalent or functional fragment thereof, any dCas9 homolog, ortholog or paralog from any organism, and any naturally occurring or engineered dCas 9 variant. Exemplary dCas9 proteins are described herein. Any suitable mutation that inactivates the Cas9 endonuclease can be used to form dCas9, such as the D10A and H840A mutations in the wild-type Streptococcus pyogenes Cas9 amino acid sequence, or the D10A and N580A mutations in the wild-type Staphylococcus aureus Cas9 amino acid sequence.

[0028] As used herein, the terms "nCas9," "Cas9 nickase," or "Cas9 nickase" refer to Cas9 or variants thereof that cleave only one strand of the target cleavage site, thereby introducing a nick in a double-stranded DNA molecule rather than generating a double-strand break. This can be achieved by introducing appropriate mutations into wild-type Cas9 to inactivate the individual activities of one of the two endonucleases of Cas9. Any suitable mutation that inactivates one of the Cas9 endonucleases while leaving the other unchanged can be used to form nCas9, such as one of the D10A or H840A mutations in the wild-type Streptococcus pyogenes Cas9 amino acid sequence, or the D10A mutation in the wild-type Staphylococcus aureus Cas9 amino acid sequence.

[0029] The term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of the nucleobase adenine. In some embodiments, adenosine deaminase catalyzes the hydrolytic deamination of adenine in deoxyribonucleic acid (DNA) to hypoxanthine. The deaminases provided herein can be from any organism, such as bacteria. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism. In some embodiments, the deaminase or deaminase domain is not naturally occurring. For example, in some embodiments, the deaminase or deaminase domain is not naturally occurring, but 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 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to a naturally occurring deaminase.

[0030] As used herein, the term "adenosine deaminase domain" refers to a domain within a base editor comprising one or more adenosine deaminase enzymes. For example, an adenosine deaminase domain may include a single adenosine deaminase enzyme. In other embodiments, the adenosine deaminase domain may include a heterodimer of a first adenosine deaminase and a second deaminase, for example, wherein the two deaminases are connected by a linker. Provided herein is an adenosine deaminase (e.g., an engineered adenosine deaminase) that can include an enzyme that converts adenosine (A) in DNA into inosine (I). This adenosine deaminase can cause the conversion of A: T to G: C base pairs. In some embodiments, the adenosine deaminase is derived from bacteria, such as Mangrovibacter sp. (e.g., Mangrovibacter sp. MFB070), Zophobihabitans entericus, or Erwinia sp. (e.g., Erwinia sp. 198).

[0031] As used herein, the term "DNA binding protein" or "DNA binding protein domain" refers to any protein that localizes and binds to a specific target DNA nucleotide sequence (e.g., a genomic locus). This term includes RNA programmable proteins that bind (e.g., form a complex) with one or more nucleic acid molecules (i.e., including, for example, guide RNAs in the case of Cas systems) that guide or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a DNA sequence) that is complementary to the one or more nucleic acid molecules (or a portion or region thereof). Exemplary RNA programmable proteins are CRISPR-Cas9 proteins, and Cas9 equivalents, homologs, orthologs, or paralogs, whether naturally occurring or non-naturally occurring (e.g., engineered or modified), and can include Cas9 equivalents from any type of CRISPR system (e.g., type II, type V, type VI), including Cpf1 (type V CRISPR-Cas system), C2c1 (type V CRISPR-Cas system), C2c2 (type VI CRISPR-Cas system), C2c3 (type V CRISPR-Cas system), dCas9, GeoCas9, CjCas9, Cas12a, Cas12b, Cas12c, Cas12d, Cas12f, Cas12g, Cas12h, Cas12i, Cas12m, Cas13d, Cas14, Argonaute, and nCas9.

[0032] The term "effective amount" as used herein refers to the amount of a bioactive agent sufficient to induce a desired biological response. For example, in some embodiments, the effective amount of a composition may refer to the amount of a composition sufficient to edit a target site of a nucleotide sequence (e.g., a genome). In some embodiments, the effective amount of a composition provided herein, for example, a composition comprising a programmable DNA binding protein domain, a deaminase domain, and a gRNA that is inactive for a nuclease, may refer to the amount of a composition sufficient to induce editing of a target site specifically bound and edited by a base editor. In some embodiments, the effective amount of a composition provided herein may refer to the amount of a composition sufficient to induce editing, having the following characteristics: >50% product purity, <5% indel, and an editing window of 2-8 nucleotides. As will be appreciated by those skilled in the art, the effective amount of an agent (e.g., a composition or a base editor-gRNA complex) may vary according to various factors, including but not limited to the desired biological response, the specific allele to be edited, the genome or target site, the cell or tissue to be targeted, and / or the base editor used.

[0033] The term "fusion protein" as used herein refers to a hybrid polypeptide comprising the protein domains of at least two proteins. One protein may be located at the amino terminal (N-terminal) portion or the carboxyl terminal (C-terminal) portion of the fusion protein. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising a peptide linker.

[0034] The term "linker" or "linker" as used herein refers to a chemical group or molecule that connects two molecules or domains, such as a linker that connects dCas9 and a deaminase. Typically, the linker is located between or on both sides of two groups, molecules or other domains and is interconnected by covalent bonds, thereby connecting the two. In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an organic molecule, group, polymer, or chemical domain. Chemical groups include, but are not limited to, disulfide, hydrazone, and azide domains. In some embodiments, the length of the linker is 5-100 amino acids, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150 or 150-200 amino acids in length. Longer or shorter linkers are also contemplated. In some embodiments, the linker is an XTEN linker. In some embodiments, the linker is a 32 amino acid linker consisting of glycine and serine. In other embodiments, the linker is a 30, 31, 33 or 34 amino acid linker consisting of glycine and serine.

[0035] The terms "non-naturally occurring" and "engineered" are used interchangeably to indicate the involvement of human effort and, when referring to nucleic acid molecules or polypeptides (e.g., deaminases), mean that the nucleic acid molecules or peptides are at least substantially free of at least one other component with which they are naturally associated and / or found in nature (e.g., an amino acid sequence not found in nature).

[0036] The term "programmable DNA binding protein" refers to any protein that can bind to (e.g., form a complex with) one or more nucleic acid molecules and direct or otherwise program the protein to localize to a specific target nucleotide sequence (e.g., a genomic locus) that is complementary to one or more nucleic acid molecules (or a portion or region thereof) associated with the protein, thereby causing the protein to bind to the nucleotide sequence at the specific target site. Exemplary programmable DNA binding proteins include CRISPR-Cas9 proteins and functional variants thereof.

[0037] Nuclear localization signal or sequence (NLS) is an amino acid sequence that marks, specifies or otherwise marks a protein to be imported into the nucleus by nuclear transport. Typically, the signal consists of one or more short sequences of positively charged lysine or arginine exposed on the surface of the protein. Different nuclear localization proteins may share the same NLS. NLS has a function opposite to that of nuclear export signal (NES), which targets proteins outside the nucleus. Therefore, a single nuclear localization signal can guide the entity associated with it to the nucleus of the cell. Such a sequence can be of any size and composition, for example, more than 25, 25, 15, 12, 10, 8, 7, 6, 5 or 4 amino acids.

[0038] As used herein, the term "subject" or "subject" refers to an individual organism, such as an individual mammal. In some embodiments, the subject is a human.

[0039] As used herein, the term "variant" refers to an equivalent of an object that has a change compared to the reference object but still retains at least one function or property (such as binding, interaction and / or enzymatic activity). A "variant" may be at least about 70% identical, at least about 80% identical, at least about 90% identical, at least approximately 95% identical, at least about 96% identical, at least substantially 97% identical, at least substantially 98% identical, at least 99% identical, at least 99.5% identical, or at least about 99.9% identical to a reference object (e.g., a wild-type protein). For example, a variant of Cas9 may include a Cas9 having one or more changes in amino acid residues compared to the wild-type Cas9 amino acid sequence. As another example, a variant of a deaminase may include a deaminase having one or more changes in amino acid residues compared to the wild-type deaminase amino acid sequence. These changes include chemical modifications, such as substitutions of different amino acid residues, truncations, covalently attached tags, and any other mutations. The term also includes fragments of wild-type proteins. The level or degree of retention of the property or function may be reduced or increased relative to the wild-type protein, but is generally the same or similar. Typically, variants are generally very similar and identical in many regions to the amino acid sequence of proteins described herein. Variant proteins may comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of, for example, a wild-type protein or any protein provided herein (e.g., an adenine base editor's adenosine deaminase domain), or consist of. For polynucleotide molecules, variants thereof include polynucleotide molecules that hybridize to the complement of the polynucleotide molecule under stringent hybridization conditions, wherein the stringent hybridization conditions are, for example, about 45°C, 6x sodium chloride / sodium citrate (SSC) in which the bound DNA is filtered, followed by washing once or multiple times at about 50-65°C in 0.2xSSC, 0.1% SDS.

[0040] A polypeptide having an amino acid sequence that is at least, for example, 95% "identical" to a reference amino acid sequence means that the amino acid sequence of the polypeptide may include up to 5 amino acid changes per 100 amino acids in the amino acid sequence of the polypeptide compared to the reference sequence. In other words, up to 5% of the amino acid residues in the reference sequence may be inserted, deleted, or substituted to obtain a polypeptide having an amino acid sequence that is at least 95% identical to the reference amino acid sequence. These changes in the reference sequence may occur at the amino or carboxyl terminal positions of the reference amino acid sequence, or anywhere between these terminal positions, or may be interspersed individually between residues in the reference sequence, or in one or more consecutive residues within the reference sequence.

[0041] Whether any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of a reference protein (e.g., any of the exemplary deaminases of the invention) can be routinely determined using known computer programs. A preferred method for determining the best overall match (also known as a global sequence alignment) between a query sequence (e.g., a deaminase sequence of the invention) and a subject sequence can be performed using the FASTDB computer program, which is based on the algorithm described by Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). In a sequence alignment, the query sequence and the subject sequence can be either nucleotide sequences or amino acid sequences. The results of the global sequence alignment are expressed as a percentage of identity. The preferred parameters used in the FASTDB amino acid alignment are: Matrix = PAM 0, k-tupie = 2, Mismatch Penalty = 1, Joining Penalty = 20, Randomization Group Length = 0, Cutoff Score = 1, Window Size = sequence length, Gap Penalty = 5, Gap Size Penalty = 0.05, Window Size = 500 or the length of the subject amino acid sequence, whichever is shorter. If the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, rather than internal deletions, the results must be manually corrected. This is because the FASTDB program does not take into account N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences that are truncated at the N- and C-termini, the percent identity is corrected relative to the query sequence by calculating the number of residues in the query sequence that are not matched / aligned with the corresponding subject residues at the N- and C-termini of the subject sequence as a percentage of the total bases in the query sequence. Whether a residue is matched / aligned is determined by the results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity calculated using the specified parameters by the FASTDB program to yield a final percent identity score. This final percent identity score is the score used for the purposes of the present invention. For manual adjustment of the percent identity score, only the N- and C-terminal residues of the subject sequence that are not matched / aligned with the query sequence are considered. In other words, only residue positions beyond the farthest N- and C-terminal residues of the subject sequence are queried.

[0042] As used herein, the term "wild type" is a term understood by those skilled in the art to refer to the typical form of an organism, strain, gene or trait occurring in nature, as distinguished from mutant or variant forms.

[0043] Adenosine deaminase

[0044] One aspect of the present invention provides adenosine deaminase.

[0045] In some embodiments, the present invention provides an adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 1: W23, Y36, P48, H51, L84, A106, D108, V109, K110, T111, D119, G122, H123, S146, F149, R152, H156, K157, E168 and E169, and having an amino acid sequence with about 70% to about 99.5% sequence identity to the sequence shown in SEQ ID NO: 1.

[0046] In some embodiments, the provided adenosine deaminase has an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty-three, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty-nine positions relative to the sequence set forth in SEQ ID NO: 1: W23, Y36, P48, H51, L84, A106, D108, V109, K110, T111, D119, G122, H123, S146, F149, R152, H156, K157, E168, and E169, and has an amino acid sequence that is about 70% to about 99.5% identical to the sequence set forth in SEQ ID NO: 1.

[0047] In some embodiments, the provided adenosine deaminase has an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty-three, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty-nine positions relative to the sequence set forth in SEQ ID NO: 1: W23, Y36, P48, H51, L84, A106, D108, V109, K110, T111, D119, G122, H123, S146, F149, R152, H156, K157, E168, and E169, and its amino acid sequence is identical to that of SEQ ID NO: The sequence shown in NO:1 has a sequence identity of about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75%.

[0048] In some embodiments, the amino acid sequence of the adenosine deaminase provided has about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75% sequence identity to the sequence shown in SEQ ID NO: 1, and comprises one or more substitutions selected from the group consisting of: W2 3R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0049] In some embodiments, the amino acid sequence of the adenosine deaminase provided is about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75% sequence identity to the sequence shown in SEQ ID NO: 1, and comprises all of the following substitutions: W23R, W23K or W23H; Y36L, Y36 V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0050] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0051] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23K; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0052] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0053] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0054] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36V; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0055] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36I; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0056] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0057] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions selected from the group consisting of W23R, W23K, or W23H; Y36L, Y36V, Y36I, or Y36P; P48A; H51L; L84F, L84W, or L84Y; A106V, A106I, A106L, or A106P; D108N; V109S; K110R; T111R, T111K, or T111H; D119N, D119R, or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W, or H156Y; K157N; E168I; and E169N.

[0058] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51V; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0059] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions selected from the group consisting of W23R, W23K, or W23H; Y36L, Y36V, Y36I, or Y36P; P48A; H51I; L84F, L84W, or L84Y; A106V, A106I, A106L, or A106P; D108N; V109S; K110R; T111R, T111K, or T111H; D119N, D119R, or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W, or H156Y; K157N; E168I; and E169N.

[0060] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions selected from the group consisting of W23R, W23K, or W23H; Y36L, Y36V, Y36I, or Y36P; P48A; H51P; L84F, L84W, or L84Y; A106V, A106I, A106L, or A106P; D108N; V109S; K110R; T111R, T111K, or T111H; D119N, D119R, or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W, or H156Y; K157N; E168I; and E169N.

[0061] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0062] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84W; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0063] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0064] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0065] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106I; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0066] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106L; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0067] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0068] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0069] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111K; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0070] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0071] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0072] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119R; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0073] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0074] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0075] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149H; R152P; H156F, H156W or H156Y; K157N; E168I; and E169N.

[0076] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156F; K157N; E168I; and E169N.

[0077] In some embodiments, the amino acid sequence of adenosine deaminase is provided as in SEQ ID The sequence shown in NO:1 includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156W; K157N; E168I; and E169N.

[0078] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 1 that includes one, more or all of the following substitutions: W23R, W23K or W23H; Y36L, Y36V, Y36I or Y36P; P48A; H51L, H51V, H51I or H51P; L84F, L84W or L84Y; A106V, A106I, A106L or A106P; D108N; V109S; K110R; T111R, T111K or T111H; D119N, D119R or D119Q; G122N; H123Y; S146C; F149Y or F149H; R152P; H156Y; K157N; E168I; and E169N.

[0079] In some embodiments, any of the provided adenosine deaminases does not contain the F149Q substitution relative to SEQ ID NO: 1.

[0080] In some embodiments, the provided adenosine deaminase comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 10, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto, provided that the amino acid sequence of the adenosine deaminase is not the sequence set forth in SEQ ID NO: 1.

[0081] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:2, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:1.

[0082] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:3, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:3, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:1.

[0083] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:4, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:4, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:1.

[0084] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:5, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:5, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:1.

[0085] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:6, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:6, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:1.

[0086] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:7, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:1.

[0087] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:8, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:1.

[0088] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:9, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:9, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:1.

[0089] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO: 10, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO: 1.

[0090] In some embodiments, the amino acid sequence of the provided adenosine deaminase consists of, or consists essentially of, the amino acid sequence of any one of SEQ ID NOs: 2 to 10.

[0091] In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 2. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 3. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 4. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 5. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 6. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 7. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 8. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 10. In some embodiments, the present invention provides an adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 12: E22, T47, L83, A105, D107, F108, T110, D118, R121, S145, F148, R151, E154, K156, V167 and E168, and having an amino acid sequence with about 70% to about 99.5% sequence identity to the sequence shown in SEQ ID NO: 12.

[0092] In some embodiments, the provided adenosine deaminase has an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty-three, fourteen, fifteen, or sixteen of the following positions relative to SEQ ID NO: 12: E22, T47, L83, A105, D107, F108, T110, D118, R121, S145, F148, R151, E154, K156, V167, and E168, and has an amino acid sequence with about 70% to about 99.5% sequence identity to SEQ ID NO: 12.

[0093] In some embodiments, the provided adenosine deaminase has an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty-three, fourteen, fifteen, or sixteen of the following positions relative to the sequence set forth in SEQ ID NO: 12: E22, T47, L83, A105, D107, F108, T110, D118, R121, S145, F148, R151, E154, K156, V167, and E168, and its amino acid sequence is identical to that of SEQ ID NO: 12. The sequence shown in NO:12 has a sequence identity of about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75%.

[0094] In some embodiments, the amino acid sequence of the adenosine deaminase provided has about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75% sequence identity to the sequence shown in SEQ ID NO: 12, and comprises One or more substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0095] In some embodiments, the amino acid sequence of the adenosine deaminase provided is about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75% sequence identity to the sequence shown in SEQ ID NO: 12, and comprises all of the following substitutions: E22R, E22K, and E22H; T 47A; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; F108V, F108I, F108L and F108P; T110R, T110K and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K156N; V167I; and E168N.

[0096] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0097] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22K; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0098] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0099] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0100] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83W; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0101] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0102] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0103] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105I; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0104] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105L; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0105] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0106] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0107] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108I; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0108] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108L; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0109] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0110] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0111] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions: E22R, E22K and E22H; T47A; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; F108V, F108I, F108L and F108P; T110K; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K156N; V167I; and E168N.

[0112] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110H; D118N; R121N; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K156N; V167I; and E168N.

[0113] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154V; K156N; V167I; and E168N.

[0114] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154I; K156N; V167I; and E168N.

[0115] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions: E22R, E22K and E22H; T47A; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; F108V, F108I, F108L and F108P; T110R, T110K and T110H; D118N; R121N; S145C; F148Y; R151P; E154L; K156N; V167I; and E168N.

[0116] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 12 that includes one, more or all of the following substitutions selected from the group consisting of: E22R, E22K, and E22H; T47A; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; F108V, F108I, F108L, and F108P; T110R, T110K, and T110H; D118N; R121N; S145C; F148Y; R151P; E154P; K156N; V167I; and E168N.

[0117] In some embodiments, the provided adenosine deaminase comprises an amino acid sequence selected from any one of SEQ ID NOs: 13 to 16, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto, provided that the amino acid sequence of the adenosine deaminase is not the sequence set forth in SEQ ID NO: 12.

[0118] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO: 13, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO: 12.

[0119] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO: 14, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO: 12.

[0120] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 15, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO: 15, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO: 12.

[0121] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO: 16, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO: 12.

[0122] In some embodiments, the amino acid sequence of the provided adenosine deaminase consists of, or consists essentially of, the amino acid sequence of any one of SEQ ID NOs: 13 to 16.

[0123] In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 13. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 14. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 15. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 16.

[0124] In some embodiments, the present invention provides an adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 17: W22, Q35, P47, Y50, L83, A105, D107, E108, T110, D118, G121, H122, S145, F148, R151, E154, K155 and K156, and having an amino acid sequence with about 70% to about 99.5% sequence identity to the sequence shown in SEQ ID NO: 17.

[0125] In some embodiments, the provided adenosine deaminase has an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty-three, fourteen, fifteen, sixteen, seventeen, or eighteen of the following positions relative to SEQ ID NO: 17: W22, Q35, P47, Y50, L83, A105, D107, E108, T110, D118, G121, H122, S145, F148, R151, E154, K155, and K156, and has an amino acid sequence with about 70% to about 99.5% sequence identity to SEQ ID NO: 17.

[0126] In some embodiments, the provided adenosine deaminase has an amino acid substitution at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirty-three, fourteen, fifteen, sixteen, seventeen, or eighteen of the following positions relative to the sequence set forth in SEQ ID NO: 17: W22, Q35, P47, Y50, L83, A105, D107, E108, T110, D118, G121, H122, S145, F148, R151, E154, K155, and K156, and its amino acid sequence is identical to that of SEQ ID NO: 17. The sequence shown in NO:17 has a sequence identity of about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75%.

[0127] In some embodiments, the amino acid sequence of the adenosine deaminase provided has about 70% to about 99%, about 75% to about 99%, about 80% to about 99%, about 85% to about 99%, about 90% to about 99%, about 70% to about 95%, about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75% sequence identity to the sequence shown in SEQ ID NO: 17, and comprises one or more substitutions selected from the group consisting of: substitutions: W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0128] In some embodiments, the amino acid sequence of the adenosine deaminase provided is about 70% to about 90%, about 70% to about 89%, about 70% to about 88%, about 70% to about 87%, about 70% to about 86%, about 70% to about 85%, about 70% to about 84%, about 70% to about 83%, about 70% to about 82%, about 70% to about 81%, about 70% to about 80%, about 70% to about 75% sequence identity to the sequence shown in SEQ ID NO: 17, and comprises all of the following substitutions: W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35 P; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0129] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R; Q35L, Q35V, Q35I and Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0130] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22K; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0131] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22H; Q35L, Q35V, Q35I and Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0132] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35L; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0133] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35V; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0134] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35I; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0135] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0136] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0137] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50V; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0138] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50I; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0139] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0140] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35L, Q35V, Q35I and Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83F; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0141] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35L, Q35V, Q35I and Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83W; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0142] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0143] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35L, Q35V, Q35I and Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0144] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105I; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0145] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105L; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0146] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0147] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F, K155W, and K155Y; and K156N.

[0148] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35L, Q35V, Q35I and Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110K; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0149] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35L, Q35V, Q35I and Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L and E154P; K155F, K155W and K155Y; and K156N.

[0150] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V; K155F, K155W, and K155Y; and K156N.

[0151] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions: W22R, W22K and W22H; Q35L, Q35V, Q35I and Q35P; P47A; Y50L, Y50V, Y50I and Y50P; L83F, L83W and L83Y; A105V, A105I, A105L and A105P; D107N; E108S; T110R, T110K and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154I; K155F, K155W and K155Y; and K156N.

[0152] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154L; K155F, K155W, and K155Y; and K156N.

[0153] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154P; K155F, K155W, and K155Y; and K156N.

[0154] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155F; and K156N.

[0155] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155W; and K156N.

[0156] In some embodiments, the amino acid sequence of the adenosine deaminase provided is a sequence shown in SEQ ID NO: 17 that includes one, more or all of the following substitutions selected from the group consisting of W22R, W22K, and W22H; Q35L, Q35V, Q35I, and Q35P; P47A; Y50L, Y50V, Y50I, and Y50P; L83F, L83W, and L83Y; A105V, A105I, A105L, and A105P; D107N; E108S; T110R, T110K, and T110H; D118N; G121N; H122Y; S145C; F148Y; R151P; E154V, E154I, E154L, and E154P; K155Y; and K156N.

[0157] In some embodiments, the provided adenosine deaminase comprises an amino acid sequence selected from any one of SEQ ID NOs: 18 to 21, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity thereto, provided that the amino acid sequence of the adenosine deaminase is not the sequence set forth in SEQ ID NO: 17.

[0158] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO: 18, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO: 17.

[0159] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO: 19, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO: 17.

[0160] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:20, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:20, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:17.

[0161] In some embodiments, the provided adenosine deaminase comprises the amino acid sequence of SEQ ID NO:21, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the amino acid sequence of SEQ ID NO:21, provided that the amino acid sequence of the adenosine deaminase is not the sequence of SEQ ID NO:17.

[0162] In some embodiments, the amino acid sequence of the provided adenosine deaminase consists of, or consists essentially of, the amino acid sequence of any one of SEQ ID NOs: 18 to 21.

[0163] In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 18. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 19. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 20. In some embodiments, the amino acid sequence of the adenosine deaminase provided consists of, or consists essentially of, the amino acid sequence of SEQ ID NO: 21.

[0164] As those skilled in the art will appreciate, any one of the adenosine deaminase enzymes provided by the present invention may be independent, or covalently bound to other parts to form an adenosine deaminase domain, which, together with the other parts, constitutes a molecule comprising the adenosine deaminase domain, such as a base editor. The term "adenosine deaminase" as used herein is intended to include the adenosine deaminase domain form. The adenosine deaminase domain may be covalently linked to other parts by one, more or all of the N-terminal, C-terminal or any position therebetween. For example, the N-terminus of adenosine deaminase is covalently linked to the C-terminus of the nuclear localization sequence (e.g., by -NH2 of the amino acid residue at the most N-terminal end of the adenosine deaminase and -COOH of the amino acid residue at the most C-terminal end of the nuclear localization sequence to form a peptide bond), while the C-terminus of the adenosine deaminase may remain free (i.e., containing free -COOH). For another example, the C-terminus of adenosine deaminase is covalently linked to the N-terminus of the nuclear localization sequence (e.g., by -NH2 forming a peptide bond with -COOH), while the N-terminus of adenosine deaminase can remain in a free state (i.e., containing free -NH2). For another example, the N-terminus of adenosine deaminase is covalently linked to the C-terminus of the nuclear localization sequence, and the C-terminus of adenosine deaminase is covalently linked to the N-terminus of the DNA binding protein. Therefore, any one of the adenosine deaminases provided by the present invention can be included in a larger protein molecule to form an adenosine deaminase domain, which retains adenosine deaminase activity and, together with the other domains, enables the larger protein molecule to function, such as base editing function. This will be described in more detail below.

[0165] base editors

[0166] Another aspect of the present invention provides a base editor.

[0167] In some embodiments, the present invention provides a base editor comprising any one of the above adenosine deaminases of the present invention. In some embodiments, the base editor is an adenine base editor. In other embodiments, the base editor is an adenine and cytosine base editor, which comprises both adenosine deaminase and cytidine deaminase, thereby achieving simultaneous editing of adenine and cytosine. In the present invention, the base editor functions in the cell as a fusion protein.

[0168] Therefore, in some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, which is formed by any one of the above adenosine deaminases of the present invention.

[0169] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 10, 13 to 16, and 18 to 21, or an amino acid sequence having at least about 80% sequence identity with each of them, provided that the amino acid sequence of the adenosine deaminase domain is not the sequence shown in SEQ ID NO: 1, 12 or 17.

[0170] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 10, 13 to 16, and 18 to 21, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto, provided that the amino acid sequence of the adenosine deaminase domain is not the sequence shown in SEQ ID NO: 1, 12 or 17.

[0171] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 1.

[0172] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO:3 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO:3, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO:1.

[0173] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO:4, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO:1.

[0174] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO:5 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO:5, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO:1.

[0175] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO:6 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO:6, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO:1.

[0176] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO:7 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO:7, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO:1.

[0177] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO:8 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO:8, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO:1.

[0178] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO:9, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO:1.

[0179] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 10, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 1.

[0180] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 13, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 12.

[0181] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 14, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 12.

[0182] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 15, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 12.

[0183] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 16, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 12.

[0184] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 18 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 18, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 17.

[0185] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 19 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 19, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 17.

[0186] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 20 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 20, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 17.

[0187] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 21, provided that the amino acid sequence of the adenosine deaminase is not the sequence shown in SEQ ID NO: 17.

[0188] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises a heteromer of a first deaminase domain and a second deaminase domain, wherein the first deaminase domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 10, 13 to 16, and 18 to 21, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto, provided that the amino acid sequence of the first adenosine deaminase domain is not the sequence shown in SEQ ID NO: 1, 12 or 17; the second deaminase domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 10, 13 to 16, and 18 to 21, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto. The amino acid sequence shown in any one of NOs: 1, 12 and 17, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto.

[0189] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises a heteromer of a first deaminase domain and a second deaminase domain, wherein the first deaminase domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 10, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto, provided that the amino acid sequence of the first adenosine deaminase domain is not the sequence shown in SEQ ID NO: 1; the second deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto. NO:1 has an amino acid sequence with at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity.

[0190] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises a heteromer of a first deaminase domain and a second deaminase domain, wherein the first deaminase domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 13 to 16, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto, provided that the amino acid sequence of the first adenosine deaminase domain is not the sequence shown in SEQ ID NO: 12; the second deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto. NO:12 has an amino acid sequence with at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity.

[0191] In some embodiments, the present invention provides a base editor comprising an adenosine deaminase domain, wherein the adenosine deaminase domain comprises a heteromer of a first deaminase domain and a second deaminase domain, wherein the first deaminase domain comprises an amino acid sequence selected from any one of SEQ ID NOs: 18 to 21, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto, provided that the amino acid sequence of the first adenosine deaminase domain is not the sequence shown in SEQ ID NO: 17; the second deaminase domain comprises the amino acid sequence shown in SEQ ID NO: 17 or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity thereto. NO:12 has an amino acid sequence with at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity.

[0192] In some embodiments, in an embodiment of any of the above-mentioned base editors, the base editor comprises a programmable DNA binding domain. In such an embodiment, the programmable DNA binding domain can be a CRISPR-associated nuclease. Exemplary CRISPR-associated nucleases include Cas9 nucleases and any variants thereof. In various embodiments, the programmable DNA binding domain has nickase activity, i.e., only cuts one chain of the target DNA sequence (such as nCas9). In other embodiments, the programmable DNA binding domain has an inactive nuclease, for example, a "dead" protein (such as dCas9). The base editors described herein may also include Cas9 equivalents, including Cas12a, Cas12b, Cas12f, Cas12i, and Cas12m proteins. The programmable DNA binding domain used herein (e.g., SpCas9, SaCas9, SaCas9 variants, or SpCas9 variants) may also include various modifications that change / enhance its PAM specificity. The present disclosure relates to any Cas9, Cas9 variant, or Cas9 equivalent having 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%, or at least 99.9% sequence identity to a reference Cas9 sequence, such as the dCas9 and nCas9 nucleases set forth in SEQ ID NO: 22 or 23.

[0193] In some embodiments, the programmable DNA binding domain may include more than one programmable DNA binding protein. Therefore, in some embodiments, any of the above-mentioned base editors may include a first programmable DNA binding domain and a second programmable DNA binding domain. In some embodiments, The programmable DNA binding domain (or the first and second programmable DNA binding domains, respectively) comprises a first Cas homolog or variant and a second Cas homolog or variant (e.g., the first Cas variant comprises Cas9-NG, and the second Cas variant comprises Cas9-CP1041, e.g., "SpCas9-NG-CPI041"). In some embodiments, the first Cas variant comprises Cas9-NG, and the second Cas variant comprises SpCas9-VRQR.

[0194] In some embodiments, in an embodiment of any of the above-mentioned base editors, the base editor comprises one or more nuclear localization sequences (or nuclear localization signals). In some embodiments, the base editor comprises a nuclear localization sequence (NLS). In other embodiments, the base editor comprises two nuclear localization sequences. In other embodiments, each of the nuclear localization sequences may be a single-component (monopartite) or two-component (bipartite) NLS. In some embodiments, the base editor comprises two two-component nuclear localization sequences.

[0195] In some embodiments, the nuclear localization sequence comprises the nucleotide sequence or amino acid sequence set forth in any one of SEQ ID NOs: 32 to 36. In some embodiments, the nuclear localization sequence comprises the nucleotide sequence set forth in SEQ ID NO: 32. In some embodiments, the nuclear localization sequence comprises the amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the nuclear localization sequence comprises the amino acid sequence set forth in SEQ ID NO: 34. In some embodiments, the nuclear localization sequence comprises the amino acid sequence set forth in SEQ ID NO: 35. In some embodiments, the nuclear localization sequence comprises the amino acid sequence set forth in SEQ ID NO: 36.

[0196] In some embodiments, in any of the above base editor embodiments, the base editor comprises a cytosine deaminase to form a base editor (CABE) that can simultaneously edit cytosine and adenine. Exemplary cytosine deaminases include AID, APOBEC3A, APOBEC1, PmCDA1, LjCDA1, or homologs thereof.

[0197] In certain embodiments, a linker can be used to connect any peptide or peptide domain or domain of a base editor (e.g., an adenosine deaminase domain is covalently linked to a programmable DNA binding domain, which in turn is covalently linked to an NLS domain). The base editors described herein may include a linker having a length of 32 amino acids. In some embodiments, the linker can connect Cas9 to a deaminase. In some embodiments, the linker can connect dCas9 to a deaminase. In some embodiments, the linker can connect nCas9 to a deaminase. The linker may be located between two groups, molecules, or other moieties, or flanked by two groups, molecules, or other moieties, and linked to each other by covalent bonds or non-covalent interactions. In some embodiments, the linker may be a polynucleotide. In some embodiments, the linker may be a DNA linker sequence. Exemplary linkers include amino acid sequences or nucleotide sequences as shown in any one of SEQ ID NOs: 11, 26, 27, 37 to 41. In some embodiments, the base editor may include multiple linkers, each linker being the same or different.

[0198] The present disclosure further provides guide RNAs for use according to the disclosed editing methods. The present disclosure provides guide RNAs designed to identify target sequences. Such gRNAs can be designed to have guide sequences (or "spacers") that are complementary to the original spacers within the target sequence. Guide RNAs for use with one or more disclosed base editors are also provided. Such gRNAs can be designed to have guide sequences that are complementary to the original spacers within the target sequence to be edited, and have a backbone sequence that specifically interacts with the programmable DNA binding domain of any disclosed base editor (e.g., the Cas9 nickase domain of the disclosed base editor).

[0199] In some embodiments, the length of the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides. In some embodiments, each gRNA comprises at least 10 consecutive nucleotides complementary to the target sequence (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 consecutive nucleotides). The DNA sequence encoding the guide RNA can be linear or circular. In some embodiments, the nuclease Cas9 or Cas9 domain is used with one or more gRNAs.

[0200] In some embodiments, an illustrative base editor comprises the following structure:

[0201] NH2-[adenosine deaminase domain]-[programmable DNA binding domain]-COOH; or

[0202] NH2-[Programmable DNA binding domain]-[Adenosine deaminase domain]-COOH,

[0203] wherein]-[ represents an optionally present linker according to the above definition (the same applies hereinafter).

[0204] In some embodiments, a schematic structure of a base editor comprising a first and a second adenosine deaminase comprises:

[0205] NH2-[first adenosine deaminase domain]-[second adenosine deaminase domain]-[programmable DNA binding domain]-COOH;

[0206] NH2-[first adenosine deaminase domain]-[programmable DNA binding domain]-[second adenosine deaminase domain]-COOH;

[0207] NH2-[programmable DNA binding domain]-[first adenosine deaminase domain]-[second adenosine deaminase domain]-COOH;

[0208] NH2-[Second adenosine deaminase domain]-[First adenosine deaminase domain]-[Programmable DNA binding domain]-COOH;

[0209] NH2-[second adenosine deaminase domain]-[programmable DNA binding domain]-[first adenosine deaminase domain]-COOH; and

[0210] NH2-[programmable DNA-binding domain]-[second adenosine deaminase domain]-[first adenosine deaminase domain]-COOH.

[0211] In some embodiments, a schematic structure of a base editor comprising an NLS comprises:

[0212] NH2-[adenosine deaminase domain]-[programmable DNA binding domain]-[NLS]-COOH;

[0213] NH2-[programmable DNA binding domain]-[adenosine deaminase domain]-[NLS]-COOH;

[0214] NH2-[NLS]-[adenosine deaminase domain]-[programmable DNA binding domain]-COOH;

[0215] NH2-[NLS]-[programmable DNA binding domain]-[adenosine deaminase domain]-COOH;

[0216] NH2-[NLS]-[adenosine deaminase domain]-[programmable DNA binding domain]-[NLS]-COOH;

[0217] NH2-[NLS]-[programmable DNA binding domain]-[adenosine deaminase domain]-[NLS]-COOH;

[0218] NH2-[NLS]-[adenosine deaminase domain]-[programmable DNA binding domain]-[NLS]-COOH; and

[0219] NH2-[NLS]-[Programmable DNA-binding domain]-[Adenosine deaminase domain]-[NLS]-COOH.

[0220] In a specific embodiment, the schematic structure of the base editor provided by the present invention includes:

[0221] NH2-[NLS]-[MaTadA1.0]-[programmable DNA binding domain]-[NLS]-COOH;

[0222] NH2-[NLS]-[Programmable DNA binding domain]-[MaTadA1.0]-[NLS]-COOH;

[0223] NH2-[NLS]-[MaTadA1.0-1]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0224] NH2-[NLS]-[Programmable DNA binding domain]-[MaTadA1.0-1]-[NLS]-COOH;

[0225] NH2-[NLS]-[MaTadA1.0-2]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0226] NH2-[NLS]-[Programmable DNA binding domain]-[MaTadA1.0-2]-[NLS]-COOH;

[0227] NH2-[NLS]-[MaTadA1.0-3]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0228] NH2-[NLS]-[Programmable DNA binding domain]-[MaTadA1.0-3]-[NLS]-COOH;

[0229] NH2-[NLS]-[MaTadA1.1]-[programmable DNA binding domain]-[NLS]-COOH;

[0230] NH2-[NLS]-[Programmable DNA-binding domain]-[MaTadA1.1]-[NLS]-COOH;

[0231] NH2-[NLS]-[MaTadA1.2]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0232] NH2-[NLS]-[Programmable DNA binding domain]-[MaTadA1.2]-[NLS]-COOH;

[0233] NH2-[NLS]-[MaTadA1.3]-[programmable DNA binding domain]-[NLS]-COOH;

[0234] NH2-[NLS]-[Programmable DNA-binding domain]-[MaTadA1.3]-[NLS]-COOH;

[0235] NH2-[NLS]-[MaTadA1.4]-[programmable DNA binding domain]-[NLS]-COOH;

[0236] NH2-[NLS]-[Programmable DNA-binding domain]-[MaTadA1.4]-[NLS]-COOH;

[0237] NH2-[NLS]-[MaTadA1.5]-[Programmable DNA binding domain]-[NLS]-COOH; or

[0238] NH2-[NLS]-[Programmable DNA-binding domain]-[MaTadA1.5]-[NLS]-COOH.

[0239] In a specific embodiment, the schematic structure of the base editor provided by the present invention includes:

[0240] NH2-[NLS]-[ZoTadA1.0]-[Programmable DNA binding domain]-[NLS]-COOH;

[0241] NH2-[NLS]-[Programmable DNA binding domain]-[ZoTada1.0]-[NLS]-COOH;

[0242] NH2-[NLS]-[ZoTadA1.0-1]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0243] NH2-[NLS]-[Programmable DNA-binding domain]-[ZoTadA1.0-1]-[NLS]-COOH;

[0244] NH2-[NLS]-[ZoTadA1.0-2]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0245] NH2-[NLS]-[Programmable DNA-binding domain]-[ZoTadA1.0-2]-[NLS]-COOH;

[0246] NH2-[NLS]-[ZoTadA1.0-3]-[Programmable DNA binding domain]-[NLS]-COOH; or

[0247] NH2-[NLS]-[Programmable DNA-binding domain]-[ZoTadA1.0-3]-[NLS]-COOH.

[0248] In a specific embodiment, the schematic structure of the base editor provided by the present invention includes:

[0249] NH2-[NLS]-[ErTadA1.0]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0250] NH2-[NLS]-[Programmable DNA-binding domain]-[ErTada1.0]-[NLS]-COOH;

[0251] NH2-[NLS]-[ErTadA1.0-1]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0252] NH2-[NLS]-[Programmable DNA-binding domain]-[ErTadA1.0-1]-[NLS]-COOH;

[0253] NH2-[NLS]-[ErTadA1.0-2]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0254] NH2-[NLS]-[Programmable DNA-binding domain]-[ErTadA1.0-2]-[NLS]-COOH;

[0255] NH2-[NLS]-[ErTadA1.0-3]-[Programmable DNA binding domain]-[NLS]-COOH; or

[0256] NH2-[NLS]-[Programmable DNA-binding domain]-[ErTadA1.0-3]-[NLS]-COOH.

[0257] In a specific embodiment, the schematic structure of the base editor provided by the present invention includes:

[0258] NH2-[NLS]-[MaTadA-WT]-[MaTadA1.0]-[programmable DNA binding domain]-[NLS]-COOH;

[0259] NH2-[NLS]-[MaTadA1.0]-[MaTadA-WT]-[programmable DNA binding domain]-[NLS]-COOH;

[0260] NH2-[NLS]-[Programmable DNA-binding domain]-[MaTadA1.0]-[MaTadA-WT]-[NLS]-COOH;

[0261] NH2-[NLS]-[Programmable DNA-binding domain]-[MaTadA-WT]-[MaTadA1.0]-[NLS]-COOH;

[0262] NH2-[NLS]-[MaTadA1.0]-[Programmable DNA-binding domain]-[MaTadA-WT]-[NLS]-COOH;

[0263] NH2-[NLS]-[MaTadA-WT]-[MaTadA1.1]-[programmable DNA binding domain]-[NLS]-COOH;

[0264] NH2-[NLS]-[MaTadA1.1]-[MaTadA-WT]-[programmable DNA binding domain]-[NLS]-COOH;

[0265] NH2-[NLS]-[Programmable DNA-binding domain]-[MaTadA1.1]-[MaTadA-WT]-[NLS]-COOH;

[0266] NH2-[NLS]-[Programmable DNA-binding domain]-[MaTadA-WT]-[MaTadA1.1]-[NLS]-COOH;

[0267] NH2-[NLS]-[MaTadA1.1]-[Programmable DNA-binding domain]-[MaTadA-WT]-[NLS]-COOH;

[0268] NH2-[NLS]-[ZoTadA-WT]-[ZoTadA1.0]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0269] NH2-[NLS]-[ZoTadA1.0]-[ZoTadA-WT]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0270] NH2-[NLS]-[Programmable DNA-binding domain]-[ZoTadA1.0]-[ZoTadA-WT]-[NLS]-COOH;

[0271] NH2-[NLS]-[Programmable DNA-binding domain]-[ZoTadA-WT]-[ZoTadA1.0]-[NLS]-COOH;

[0272] NH2-[NLS]-[ZoTadA1.0-1]-[Programmable DNA-binding domain]-[ZoTadA-WT]-[NLS]-COOH;

[0273] NH2-[NLS]-[ErTadA-WT]-[ErTadA1.0]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0274] NH2-[NLS]-[ErTadA1.0]-[ErTadA-WT]-[Programmable DNA-binding domain]-[NLS]-COOH;

[0275] NH2-[NLS]-[Programmable DNA-binding domain]-[ErTadA1.0]-[ErTadA-WT]-[NLS]-COOH;

[0276] NH2-[NLS]-[Programmable DNA-binding domain]-[ErTadA-WT]-[ErTadA1.0]-[NLS]-COOH; or

[0277] NH2-[NLS]-[ErTadA1.0-1]-[Programmable DNA-binding domain]-[ErTadA-WT]-[NLS]-COOH.

[0278] In a specific embodiment, the schematic structure of the base editor provided by the present invention includes:

[0279] NH2-[NLS]-[MaTadA1.0]-[dCas9 / nCas9]-[NLS]-COOH;

[0280] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.0]-[NLS]-COOH;

[0281] NH2-[NLS]-[MaTadA1.0-1]-[dCas9 / nCas9]-[NLS]-COOH;

[0282] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.0-1]-[NLS]-COOH;

[0283] NH2-[NLS]-[MaTadA1.0-2]-[dCas9 / nCas9]-[NLS]-COOH;

[0284] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.0-2]-[NLS]-COOH;

[0285] NH2-[NLS]-[MaTadA1.0-3]-[dCas9 / nCas9]-[NLS]-COOH;

[0286] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.0-3]-[NLS]-COOH;

[0287] NH2-[NLS]-[MaTadA1.1]-[dCas9 / nCas9]-[NLS]-COOH;

[0288] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.1]-[NLS]-COOH;

[0289] NH2-[NLS]-[MaTadA1.2]-[dCas9 / nCas9]-[NLS]-COOH;

[0290] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.2]-[NLS]-COOH;

[0291] NH2-[NLS]-[MaTadA1.3]-[dCas9 / nCas9]-[NLS]-COOH;

[0292] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.3]-[NLS]-COOH;

[0293] NH2-[NLS]-[MaTadA1.4]-[dCas9 / nCas9]-[NLS]-COOH;

[0294] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.4]-[NLS]-COOH;

[0295] NH2-[NLS]-[MaTadA1.5]-[dCas9 / nCas9]-[NLS]-COOH; or

[0296] NH2-[NLS]-[dCas9 / nCas9]-[MaTadA1.5]-[NLS]-COOH.

[0297] In a specific embodiment, the schematic structure of the base editor provided by the present invention includes:

[0298] NH2-[NLS]-[ZoTadA1.0]-[dCas9 / nCas9]-[NLS]-COOH;

[0299] NH2-[NLS]-[dCas9 / nCas9]-[ZoTadA1.0]-[NLS]-COOH;

[0300] NH2-[NLS]-[ZoTadA1.0-1]-[dCas9 / nCas9]-[NLS]-COOH;

[0301] NH2-[NLS]-[dCas9 / nCas9]-[ZoTadA1.0-1]-[NLS]-COOH;

[0302] NH2-[NLS]-[ZoTadA1.0-2]-[dCas9 / nCas9]-[NLS]-COOH;

[0303] NH2-[NLS]-[dCas9 / nCas9]-[ZoTadA1.0-2]-[NLS]-COOH;

[0304] NH2-[NLS]-[ZoTadA1.00-3]-[dCas9 / nCas9]-[NLS]-COOH; or

[0305] NH2-[NLS]-[dCas9 / nCas9]-[ZoTadA1.0-3]-[NLS]-COOH.

[0306] In a specific embodiment, the schematic structure of the base editor provided by the present invention includes:

[0307] NH2-[NLS]-[ErTadA1.0]-[dCas9 / nCas9]-[NLS]-COOH;

[0308] NH2-[NLS]-[dCas9 / nCas9]-[ErTadA1.0]-[NLS]-COOH;

[0309] NH2-[NLS]-[ErTadA1.0-1]-[dCas9 / nCas9]-[NLS]-COOH;

[0310] NH2-[NLS]-[dCas9 / nCas9]-[ErTadA1.0-1]-[NLS]-COOH;

[0311] NH2-[NLS]-[ErTadA1.0-2]-[dCas9 / nCas9]-[NLS]-COOH;

[0312] NH2-[NLS]-[dCas9 / nCas9]-[ErTadA1.0-2]-[NLS]-COOH;

[0313] NH2-[NLS]-[ErTadA1.00-3]-[dCas9 / nCas9]-[NLS]-COOH; or

[0314] NH2-[NLS]-[dCas9 / nCas9]-[ErTadA1.0-3]-[NLS]-COOH.

[0315] In a specific embodiment, the base editor provided by the present invention may comprise an amino acid sequence as shown in any one of SEQ ID NOs: 46 to 54, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NOs: 46 to 54, or essentially consisting of any one of the amino acid sequences.

[0316] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:46, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:46, or is essentially composed thereof.

[0317] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:47, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:47, or is essentially composed thereof.

[0318] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:48, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:48, or is essentially composed thereof.

[0319] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:49, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:49, or is essentially composed thereof.

[0320] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:50, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:50, or is essentially composed thereof.

[0321] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:51, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:51, or is essentially composed of the same.

[0322] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:52, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:52, or is essentially composed thereof.

[0323] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:53, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:53, or is essentially composed thereof.

[0324] In some embodiments, the base editor provided by the present invention comprises an amino acid sequence as shown in SEQ ID NO:54, or an amino acid sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity with the amino acid sequence of SEQ ID NO:54, or is essentially composed thereof.

[0325] Polynucleotides, vectors, cells

[0326] Another aspect of the present invention provides a polynucleotide encoding the adenosine deaminase provided by the present invention or a base editor comprising the adenosine deaminase.

[0327] In some embodiments, the polynucleotide encoding adenosine deaminase is a DNA or RNA sequence. In some embodiments, the polynucleotide is codon-optimized, for example, for expression in eukaryotic cells (eg, human cells).

[0328] Exemplary polynucleotide sequences encoding adenosine deaminase of the present invention include the nucleic acid sequences of any one of SEQ ID NOs: 24, 25, 28 to 31.

[0329] Another aspect of the present invention provides a vector comprising any of the above-mentioned polynucleotides, in particular a polynucleotide encoding any one of the base editors of the present invention. The vector can be designed to clone and / or express the base editor of the present invention. The vector can also be designed to transfect the adenine base editor of the present invention into one or more cells, such as target lesion eukaryotic cells, for treatment with the base editors and methods of the present invention. The vector can be designed to express base editing transcripts (eg, nucleic acid transcripts, proteins, or enzymes) in prokaryotes or eukaryotic cells. For example, base editor transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, plant cells, or mammalian cells. Alternatively, expression vectors encoding one or more base editors described herein can be transcribed and translated in vitro, for example, using T7 promoter regulatory sequences and T7 polymerase.

[0330] In some embodiments, prokaryotes are used to amplify a copy of a vector that is introduced into a eukaryotic cell, or to produce an intermediate vector (such as an amplification plasmid as a part of a viral vector packaging system) that is introduced into a eukaryotic cell. In some embodiments, prokaryotes are used to amplify a copy of a vector and express one or more nucleic acids, such as provide a source of one or more proteins to be delivered to a host cell or a host organism. Protein expression in prokaryotes is usually carried out in escherichia coli using a vector containing a constitutive or inducible promoter.

[0331] In some embodiments, the vector can drive expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 and pMT2PC. When used in mammalian cells, the control functions of the expression vector are generally provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, simian virus 40, and other promoters known in the art.

[0332] Some embodiments of the present invention provide cells comprising any base editor or complex provided herein. In some embodiments, the cell comprises a nucleotide construct encoding any base editor provided herein. In some embodiments, the cell includes any nucleotide or vector provided herein. In some embodiments, the cell is a stem cell. In some embodiments, the cell is a human stem cell, such as a human stem and progenitor cell (HSPC). In some embodiments, the cell is an activated (e.g., plerixafor activated) peripheral blood HSPC.

[0333] In some embodiments, host cells are transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, cells are naturally transfected in a subject. In some embodiments, the transfected cells are taken from a subject. In some embodiments, the cells are derived from cells taken from a subject, such as a cell line. A variety of cell lines for tissue culture are known in the art. In some embodiments, cells are removed from a subject and contacted in vitro with any disclosed base editor, complex, vector, or polynucleotide.

[0334] Pharmaceutical compositions, methods of treatment, and uses

[0335] Another aspect of the present invention provides a method for treating a subject diagnosed with a disease associated with or caused by a point mutation, which can be corrected by a base editor provided herein. For example, in some embodiments, a method is provided, comprising administering an effective amount of an adenosine base editor to a subject suffering from such a disease (e.g., a cancer associated with a point mutation as described above), which corrects the point mutation or introduces an inactivating mutation into a disease-associated gene. In some embodiments, the disease is a proliferative disease. In some embodiments, the disease is a hereditary disease. In some embodiments, the disease is a neoplastic disease. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a lysosomal storage disease. Other diseases that can be treated by correcting point mutations or introducing inactivating mutations into disease-associated genes will be known to those skilled in the art.

[0336] In some embodiments, deamination of the mutated A results in a codon encoding a wild-type amino acid. In some embodiments, the contacting is performed in a subject. In some embodiments, the subject has or has been diagnosed with a disease or condition. In some embodiments, the disease or condition is a hemoglobinopathy. In some embodiments, the disease or condition is sickle cell disease. In some embodiments, the disease or condition is thalassemia. In some embodiments, the disease or condition is glycogen storage disease type 1A associated with the R83C mutation in the glucose-6-phosphatase-alpha (G6PC) enzyme, and Stargardt macular dystrophy associated with the G1961E mutation in the ATP-binding cassette subfamily A, member 4 (ABCA4) protein. In some embodiments, the disease or condition is phenylketonuria, von Willebrand disease (vWD), a neoplastic disease associated with mutant PTEN or BRCA1, or Li-Fraumeni syndrome.

[0337] Some embodiments provide methods using base editors provided herein. In some embodiments, base editors are used to introduce point mutations into nucleic acids by deaminating target nuclear bases (e.g., A bases). In some embodiments, the deamination of target nuclear bases leads to the correction of genetic defects, such as correction of point mutations that cause loss of function in gene products. In some embodiments, provided herein are methods for introducing inactivation point mutations into genes or alleles encoding gene products related to a disease or disorder. For example, in some embodiments, provided herein are methods for introducing inactivation point mutations into oncogenes using base editors (e.g., in the treatment of proliferative diseases). In some embodiments, inactivation mutations can produce premature stop codons in the coding sequence, which result in the expression of truncated gene products (e.g., truncated proteins lacking full-length protein function).

[0338] In some embodiments, the purpose of the methods provided herein is to restore the function of dysfunctional genes by genome editing. The base editors provided herein can be validated in vitro for human treatment based on gene editing, for example, by correcting disease-associated mutations in human cell culture. Those skilled in the art will understand that the base editors provided herein can be used to correct any single-point G to A or C to T mutation.

[0339] Other aspects of the present invention relate to pharmaceutical compositions comprising any of the adenosine deaminase, base editor, or base editor-gRNA complex described herein. Other aspects of the present invention relate to pharmaceutical compositions comprising any of the polynucleotides or vectors comprising nucleic acid fragments encoding the adenosine deaminase, base editor, or base editor-gRNA complex described herein.

[0340] In some embodiments, any base editor, gRNA, and / or complex described herein is provided as part of a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any base editor provided herein. In some embodiments, the pharmaceutical composition comprises any complex provided herein. In some embodiments, the pharmaceutical composition comprises gRNA, a base editor, and a pharmaceutically acceptable excipient. The pharmaceutical composition may optionally comprise one or more additional therapeutically active substances.

[0341] In some embodiments, the compositions provided herein are formulated for delivery to a subject, e.g., to a human subject to achieve targeted genome modification within the subject. In some embodiments, cells are obtained from the subject and contacted with any pharmaceutical composition provided herein. In some embodiments, cells removed from the subject and contacted ex vivo with the pharmaceutical composition are reintroduced into the subject, optionally after the desired genome modification is achieved or detected in the cell.

[0342] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. Generally, such preparation methods include combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single-dose or multi-dose units.

[0343] In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, for example, for gene editing. Suitable routes of administration of the pharmaceutical compositions described herein include, but are not limited to, topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intramental, intracochlear, transtympanic, intraauricular, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseous, periocular, intratumoral, intracerebral, and intracerebroventricular administration.

[0344] In various embodiments, base editor constructs (including split constructs) can be designed for delivery in one or more rAAV vectors. The rAAV related to any method and composition provided herein can be any serotype, including any derivative or pseudotype (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 2 / 1, 2 / 5, 2 / 8, 2 / 9, 3 / 1, 3 / 5, 3 / 8 or 3 / 9). rAAV can include a genetic load to be delivered to a cell (i.e., a recombinant nucleic acid vector expressing a gene of interest, such as a full base or split base editor carried into a cell by rAAV). rAAV can be chimeric. As used herein, the serotype of rAAV refers to the serotype of the recombinant viral capsid protein. Non-limiting examples of derivatives and pseudotypes include rAAV2 / 1, rAAV2 / 5, rAAV2 / 8, rAAV2 / 9, AAV2-AAV3 hybrid, AAVrh.10, AAVrh.74, AAVhu.14, AAV3a / 3b, AAVrh32.33, AAV-HSC15, AAV-HSC17, AAVhu.37, AAVrh.8, CHt-P6, AAV2.5, AAV6.2, AAV2i8, AAV-HSC15 / 17, AAVM41, AAV9.45, AAV6(Y445F / Y731F), AAV2.5T, AAV-HAE1 / 2, AAV32 / 83, AAVShH10, AAV2(Y->F), AAV 8(Y733F), AAV2.15, AAV2.4, AAVM41 and AAVr3.45.

[0345] In various embodiments, the disclosed editing methods produce at least about 35%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or 99% on-target DNA base editing efficiency at the target nucleobase pair. The contacting step can result in a DNA base editing efficiency of at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or 75%. In particular, the contacting step results in a target-based editing efficiency greater than 75%. In certain embodiments, a base editing efficiency of 99% can be achieved.

[0346] In some embodiments, the editing methods of the disclosed base editors result in an actual or average off-target DNA editing frequency of about 2.0% or less, 1.75% or less, 1.5% or less, 1.2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.75% or less, 0.7% or less, 0.65% or less, or 0.6% or less in some embodiments, the disclosed editing methods produce an actual or average off-target DNA editing frequency of 0.5%, less than 0.5%, less than 0.4%, less than 0.35%, less than 0.3%, less than 0.25%, less than 0.2%, or less than 0.1%.

[0347] In some embodiments, the base pair to be edited is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides upstream of the PAM site. In some embodiments, the base pair to be edited is downstream of the PAM site. In some embodiments, the base pair to be edited is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides downstream of the PAM site. In some embodiments, the method does not require an orthodox (e.g., NGG) PAM site. In some embodiments, the target area includes a target window, wherein the target window includes a target core base pair. In some embodiments, the target window includes 1-10 nucleotides. In some embodiments, the target window is 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1 nucleotide in length. In some embodiments, the target window is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the base pair to be edited is within the target window. In some embodiments, the method is performed using any base editor provided herein. In some embodiments, the target window is a deamination window.

[0348] In some embodiments, the adenine deaminase of the adenine base editor has a high editing efficiency for adenine A at position 3, 4, 5, 6, 7, 8, or 9 of the active window, and an even higher editing efficiency for adenine A at position 3, 4, 5, or 6. In some embodiments, the editing window of MaTadA1.0, ZoTadA1.0, and ErTadA1.0 is positions 3-9 (narrower editing window, higher specificity), more preferably positions 4-8; among them, MaTadA1.0 has the highest editing efficiency in eukaryotes.

[0349] Another aspect of the present invention provides the use of an adenosine deaminase, base editor, polynucleotide, vector, complex, cell or pharmaceutical composition provided herein in the preparation of a method for treating a disease mediated by a nucleobase A mutation. Another aspect of the present invention also provides an adenosine deaminase, base editor, polynucleotide, vector, complex, cell or pharmaceutical composition provided herein for treating a disease mediated by a nucleobase A mutation. The disease mediated by a nucleobase A mutation can be any of the treatable diseases mentioned above.

[0350] Sequence Listing

[0351] Example

[0352] Example 1. Verification of the deamination effect of adenosine deaminase in prokaryotic cells (acting on the start codon of ccdB)

[0353] 1.1 Construction of multiple base editor plasmids for experimental and control groups

[0354] Experimental group base editor plasmids: There are two types. One is to construct sgRNA-ccdB1 containing a promoter (which can target the start codon of the antisense chain of the ccdB toxin gene, the sequence is 5'-CTGCATTTATGTCAGACTTG-3', SEQ ID NO: 42) in a commercial vector of the ccdB toxin gene containing an arabinose-inducible promoter. The ccdB and sgRNA were constructed to obtain plasmid A, marked as pBR322-ccdB-sgRNA-KanaR-ATG-1-gRNA1 (Figure 16A). The other is to construct the adenine deaminase (MaTadA1.0, ZoTadA1.0, ErTadA1.0) and dCas9 provided by the present invention into a commercial vector through a linker to recombinantly form adenosine deaminase-dCas9 recombinant plasmid B, which is marked as MaTadA1.0-dCas9, ZoTadA1.0-dCas9 and ErTadA1.0-dCas9, respectively (Figures 16B to D).

[0355] Control group base editor plasmid: According to the above method, two plasmids were constructed. One is to construct the existing conventional adenine deaminase TadA-8e and dCas9 through a linker in a commercial vector to recombinantly form the TadA-8e-dCas9 recombinant plasmid C, marked as TadA-8e-dCas9 (Figure 17A); the other is to construct the unmutated wild-type protein (MaTadA-WT, ZoTadA-WT or ErTadA-WT) and dCas9 through a linker in a commercial vector to recombinantly form the negative control plasmid D, marked as MaTadA-WT-dCas9, ZoTadA-WT-dCas9 and ErTadA-WT-dCas9, respectively (Figures 17A to D).

[0356] 1.2 Verification of the deamination activity of the above base editors in prokaryotic systems

[0357] The ccdB toxicity gene used in this experiment is used to detect the deamination activity of adenosine deaminase. The principle is that the expression of the ccdB toxicity gene is regulated by the AraBAD operon: when the culture environment contains a large amount of glucose (final concentration 100mM), the expression of ccdB is inhibited; when the culture environment contains arabinose, ccdB is induced to express (the higher the arabinose concentration, the higher the expression level). After induced expression of ccdB, it will interact with DNA helicase and destroy the structure of the host double-stranded DNA.

[0358] If adenine deaminase (MaTadA1.0, ZoTadA1.0, ErTadA1.0) has adenosine deamination activity, dCas9 targets and binds to the start codon region of the ccdB gene under the action of sgRNA. Under the deamination action of adenosine deaminase, the start codon of the antisense strand of the ccdB gene is edited (i.e., mutated from the original TAC to TGC), causing the start codon of the sense strand of the ccdB gene to be edited from ATG to ACG, causing the expression of the ccdB gene to shift, and failing to induce correct expression. If the ccdB is expressed, the protein expressed by the recombinant plasmid loses the ability to destroy the host double-stranded DNA structure, and the host bacteria can reproduce normally; if the adenine deaminase (MaTadA1.0, ZoTadA1.0, ErTadA1.0) does not have adenosine deamination activity, the base editor cannot work normally, and the host bacteria can induce the expression of normal ccdB protein, which will interact with the DNA helicase, destroy the structure of the host double-stranded DNA, and the host bacteria will be killed. Whether the base editor has a deamination effect can be judged by whether the Escherichia coli grows.

[0359] The experimental protocol is to transform the above-mentioned recombinant plasmid pBR322-ccdB-sgRNA-KanaR-ATG-1-gRNA1 into Escherichia coli competent cells to form a stable competent strain, and then respectively transfer MaTadA1.0-dCas9, ZoTadA1.0-dCas9, ErTadA1.0-dCas9, TadA-8e-dCas9, MaTadA-WT-dCas9, ZoTadA-WT-dCas9 or ErTadA-WT-dCas9 into the stable competent strain containing the plasmid. Escherichia coli is then spread onto LB solid medium containing 100nM glucose and different concentrations of arabinose (0nM, 20nM and 40nM) and antibiotics (kanamycin), cultured at 37°C overnight, and then observed whether Escherichia coli can grow on the solid medium. The results are shown in Figures 1 and 3A. The results showed that the adenine deaminases (MaTadA1.0, ZoTadA1.0, ErTadA1.0) and TadA-8e of the present invention had an editing effect on the start codon of the ccdB gene. Escherichia coli grew normally in all groups under the conditions of adding glucose but not adding arabinose; the dCas9 control group, MaTadA-WT-dCas9 group, ZoTadA-WT-dCas9 group and ErTadA-WT-dCas9 group did not grow under the conditions of adding different concentrations of arabinose and glucose, indicating that the base editor in E. coli did not work, and arabinose induced the normal expression of ccdB protein, leading to the death of E. coli; the TadA-8e-dCas9 group, MaTadA1.0-dCas9 group, ZoTadA1.0-dCas9 group and ErTadA1.0-dCas9 group could all grow under the conditions of adding different concentrations of arabinose and adding glucose, indicating that the adenosine deaminase of the base editor has a deamination effect, resulting in a frameshift during ccdB expression, and the normal ccdB protein is not produced in the bacteria, resulting in the survival of E. coli.

[0360] Example 2. Functional verification of other adenine deaminases in prokaryotic cells

[0361] According to the method of Example 1 above, the base editing effect of adenine deaminase MaTadA1.0-1, MaTadA1.0-2, MaTadA1.0-3, ZoTadA1.0-1, ZoTadA1.0-2, ZoTadA1.0-3, ErTadA1.0-1, ErTadA1.0-2 and ErTadA1.0-3 and ABE constructed with dCas9 (editing the start codon region in prokaryotic cells) was verified, and the results are shown in Figures 5A to C.

[0362] Example 3. Comparison of editing site efficiency between adenine deaminase MaTadA1.0 and TadA-8e in prokaryotic systems (acting on the stop codon of ccdB)

[0363] 3.1 Construction of base editor plasmids in the experimental and control groups

[0364] Experimental group base editor plasmids: There are two types. One is to construct the sgRNA-ccdB2 containing the promoter (which can target the stop codon of the antisense strand of the ccdB toxin gene, the sequence is 5'-atatagctaagatgtcacgg-3', SEQ ID NO: 43) in a commercial vector containing the ccdB toxin gene with an arabinose-inducible promoter. The ccdB and sgRNA construct plasmid E, labeled pBR322-CcdB-sgRNA-KanaR-TAG-gRNA1 (Figure 18). The other is MaTadA1.0-dCas9 from step 1.1 of Example 1 (Figure 16B).

[0365] Control group base editor plasmid: TadA-8e-dCas9 constructed in step 1.1 of Example 1 above ( Figure 17A ).

[0366] 3.2 Verification of the deamination effect of the above-mentioned base editor on adenine A in a prokaryotic system (acting on the stop codon of ccdB)

[0367] This experiment used the ccdB toxicity gene to detect the deamination activity of adenosine deaminase. The principle is that the main active domain of ccdB is located at the amino acid WGI at the end of its peptide chain. The above experiments demonstrated that the adenine deaminase MaTadA1.0 has adenosine deamination activity. Under the action of sgRNA, dCas9 targets the stop codon region of the ccdB gene. Under the deamination action of adenosine deaminase, the stop codon of the antisense strand of the ccdB gene is edited (i.e., mutated from the original ATC to GTC), resulting in the stop codon of the sense strand of the ccdB gene being edited from TAG to CAG. This causes a frameshift in the expression of the ccdB gene, preventing proper termination of expression and expression of the correct ccdB protein. The protein expressed by the recombinant plasmid loses the ability to disrupt the host double-stranded DNA structure, allowing the host bacteria to reproduce normally. The growth of E. coli can be used to determine whether the base editor has a deamination effect.

[0368] The experimental protocol involved transforming the recombinant plasmid pBR322-CcdB-sgRNA-KanaR-TAG-gRNA1 into competent E. coli cells to form a stable competent strain. MaTadA1.0-dCas9 and TadA-8e-dCas9 were then transferred into the stable competent strain containing the plasmid. E. coli were then plated onto LB solid medium containing antibiotics supplemented with 100 nM glucose and varying concentrations of arabinose (0 nM, 20 nM, and 40 nM), cultured overnight at 37°C, and observed to see if the E. coli could grow on the solid medium. The results are shown in Figures 2 and 4. All groups grew normally under the conditions of no arabinose and glucose addition. The dCas9 control group and the MaTadA-WT-dCas9 group did not grow under the conditions of adding different concentrations of arabinose and adding glucose, indicating that the base editor in E. coli did not work, and arabinose induced the normal expression of ccdB protein, leading to the death of E. coli. The TadA-8e-dCas9 group and the MaTadA1.0-dCas9 group were able to grow under the conditions of adding different concentrations of arabinose and adding glucose, indicating that the adenosine deaminase of the base editor has a deamination effect, resulting in a frameshift during ccdB expression, and no normal ccdB protein is produced in the bacteria, resulting in the survival of E. coli. The results showed that the MaTadA1.0 and TadA-8e provided by the present invention have an editing effect on the stop codon of the ccdB gene, but the editing effect is slightly weaker than that of the start codon.

[0369] Example 4. Functional verification of other adenine deaminases MaTadA1.1, MaTadA1.2, MaTadA1.3, MaTadA1.4 and MaTadA1.5 in prokaryotic cells

[0370] The base editing effect of the ABE constructed by adenine deaminases MaTadA1.1, MaTadA1.2, MaTadA1.3, MaTadA1.4, and MaTadA1.5 and dCas9 (editing the start codon region in prokaryotic cells) was verified according to the method of Example 1 above. The results are shown in Figures 1C, 1D, 3B, and 3C. As shown in Figures 3B and 3C, all the verified adenine deaminases have editing effects, among which MaTadA1.1 has the best editing effect, followed by MaTadA1.2, MaTadA1.3, and MaTadA1.5, and then MaTadA1.4 has the highest editing efficiency.

[0371] Example 5. Verification of the deamination effect of adenosine deaminase in 293T human cell line

[0372] Adenine deaminases (MaTadA1.0, ZoTadA1.0, ErTadA1.0, TadA-8e, and MaTadA-WT) were constructed into eukaryotic expression vectors containing the chicken β-actin promoter and the nCas9 gene, and containing the U6 promoter and the sgRNA sequence (the sgRNA sequence contains two segments, namely the targeting segment (also called the spacer in the context of the endogenous CRISPR system) and the protein binding segment (containing the direct repeat sequence). The target genes of the targeting segment of the sgRNA are the Hek-2 site and the ATP7B gene P992L site) to form recombinant adenine base editor vectors, which were marked as: pX330-MaTadA1.0-HeK2 (Figure 20A), pX330-ZoTadA1.0-HeK2 (Figure 20B), pX330-ErTadA1.0-HeK2 (Figure 20C), and pX330- Figure 20C), pX330-TadA-8e-HeK2 (Figure 19B), pX330-MaTadA1.0-P992L (Figure 21B), pX330-ErTadA1.0-P992L (Figure 22A), pX330-TadA-8e-P992L (Figure 21A), pX330-MaTadA-WT-P992L (Figure 22B); at the same time, a blank control plasmid without adenine deaminase sequence was constructed (the sgRNA sequence only contains the protein binding segment scaffold and does not contain the targeting segment), marked as px330 (Figure 19A). The sgRNA targeting the Hek-2 site is 5'-gaacacaaagcatagactgc-3' (SEQ The sgRNA targeting the p992L site is 5'-gcgtgagcgtggccagcccca-3' (SEQ ID NO: 45).

[0373] Then, the adenine base editor vector was transferred into human cells by lipofectamine transfection and cultured at 37°C and 5% carbon dioxide concentration for 72 hours. The DNA of all cells was extracted, and the sequence containing the target site was amplified. The PCR product was sequenced for the first generation, and the sequencing results were aligned to the corresponding genes in the human genome to measure the editing efficiency of the adenine base editor for the target gene. The results showed that the editing efficiency of the adenine base editor vector provided by the present invention for the Hek-2 site and the ATP7B gene P992L site can reach more than 50%, and some of the original sequencing data are shown in Figures 6 to 15.

[0374] Figure 6 shows the sequencing results of pX330-MaTadA1.0-HeK2 editing the HEK2 gene in 293T cells, demonstrating effective editing at positions 3, 5, and 7. The data in the table below represent the numerical results of sequencing at each nucleotide position. Values ​​below 10 represent noise. The A→G efficiency at the third position is approximately 13%, the fifth position is approximately 54%, and the seventh position is approximately 53%.

[0375] Figure 7 shows the sequencing results of pX330-ZoTadA1.0-HeK2 editing the HEK2 gene in 293T cells, demonstrating effective editing at position 5. The data in the table below represent the numerical results of sequencing at each nucleotide site. Values ​​below 10 represent noise. The efficiency of A→G at position 5 is approximately 31%.

[0376] Figure 8 shows the sequencing results of pX330-ErTadA1.0-HEK2 editing the HEK2 gene in 293T cells. The results demonstrate effective editing, with the editing window covering positions 3, 5, 7, and 8. The data in the table below represent the numerical results for sequencing at each nucleotide position. Values ​​below 10 represent noise. The efficiency of A→G at position 3 is approximately 16%, at position 5 is approximately 51%, at position 7 is approximately 40%, and at position 8 is approximately 42%. Positions 7, 8, and 9 are all A's, and the sequencing graph clearly shows a continuous peak for adenylate A, while other bases are absent. However, the software readout is affected by the continuous peaks, resulting in the display of the ratio of other bases. This is a software error. The sequencing graph is the correct indicator. As shown in the graph, the editing efficiency at position A8 is very high.

[0377] Figure 9 shows the sequencing results of pX330-TadA-8e-HeK2 editing the HEK2 gene in 293T cells, demonstrating effective editing at positions 5 and 7. The data in the table below represent the numerical results of sequencing at each nucleotide site. Values ​​below 10 represent noise. The A→G efficiency at position 5 and position 7 is approximately 41%, and the A→G efficiency at position 5 and position 7 is approximately 41%.

[0378] Figure 10 shows the sequencing results of HEK2 gene editing by the pX330 blank vector in 293T cells, and the results show no editing effect.

[0379] Figure 11 shows the sequencing results of pX330-MaTadA1.0-P992L editing the p992L gene in 293T cells, demonstrating effective editing (overlapping peaks). The efficiency of the sixth position A→G conversion is approximately 29%. Due to primer issues, the sequencing results show the antisense strand of the sgRNA (direction 3'→5'). The same applies below.

[0380] Figure 12 shows the sequencing diagram of pX330-MaTadA-WT-nCas9-P992L editing the p992L gene in 293T cells, and the results show that it has no editing effect (no overlapping peaks).

[0381] Figure 13 shows the sequencing results of pX330-ErTadA1.0-nCas9-P992L editing the p992L gene in 293T cells, showing that it has an editing effect (overlapping peaks). The efficiency of the sixth position A→G is about 31%.

[0382] Figure 14 shows the sequencing results of pX330-TadA-8e-P992L editing the p992L gene in 293T cells, demonstrating effective editing. The efficiency of A→G at position 6 is approximately 26%, which is lower than that of HEK2 gene editing.

[0383] Figure 15 shows the sequencing results of the pX330 blank vector editing the p992L gene in 293T cells, and the results showed no editing effect.

Claims

1. An adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 1: W23, Y36, P48, H51, L84, A106, D108, V109, K110, T111, D119, G122, H123, S146, F149, R152, H156, K157, E168 and E169, and having an amino acid sequence with about 70% to about 99.5% sequence identity with the sequence shown in SEQ ID NO:

1.

2. Adenosine deaminase according to claim 1, wherein: (a) the substitution of W23 is selected from the group consisting of: W23R, W23K and W23H; (b) the substitution of Y36 is selected from the group consisting of: Y36L, Y36V, Y36I and Y36P; (c) P48 is substituted with P48A; (d) a substitution of H51 is selected from the group consisting of: H51L, H51V, H51I and H51P; (e) the substitution of L84 is selected from the group consisting of: L84F, L84W and L84Y; (f) A106 is substituted with a member selected from the group consisting of A106V, A106I, A106L, and A106P; (g) D108 is substituted with D108N; (h) V109 is substituted with V109S; (i) substitution of K110 to K110R; (j) the substitution of T111 is selected from the group consisting of: T111R, T111K and T111H; (k) the substitution of D119 is selected from the group consisting of: D119N, D119R and D119Q; (l) G122 is substituted with G122N; (m) H123 is substituted with H123Y; (n) S146 is substituted with S146C; (o) the substitution of F149 is selected from the group consisting of: F149Y and F149H; (p) R152 is substituted with R152P; (q) a substitution of H156 selected from the group consisting of: H156F, H156W, and H156Y; (r) substitution of K157 to K157N; (s) E168 is substituted by E168I; or (t) The substitution of E169 is E169N.

3. The adenosine deaminase according to claim 1 or 2, wherein the adenosine deaminase comprises an amino acid sequence selected from any one of SEQ ID NOs: 2 to 10 or an amino acid sequence having at least about 80% sequence identity thereto.

4. An adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 12: E22, T47, L83, A105, D107, F108, T110, D118, R121, S145, F148, R151, E154, K156, V167 and E168, and having an amino acid sequence with about 70% to about 99.5% sequence identity with the sequence shown in SEQ ID NO:

12.

5. Adenosine deaminase according to claim 4, wherein: (a) the substitution of E22 is selected from the group consisting of: E22R, E22K and E22H; (b) T47 is substituted with T47A; (c) the substitution of L83 is selected from the group consisting of: L83F, L83W and L83Y; (d) A105 is substituted with a member selected from the group consisting of A105V, A105I, A105L, and A105P; (e) D107 is substituted with D107N; (f) a substitution of F108 selected from the group consisting of: F108V, F108I, F108L and F108P; (g) the substitution of T110 is selected from the group consisting of: T110R, T110K and T110H; (h) substitution position D118N of D118; (i) R121 is substituted with R121N; (j) S145 is substituted with S145C; (k) F148 is substituted with F148Y; (l) R151 is substituted with R151P; (m) a substitution of E154 selected from the group consisting of: E154V, E154I, E154L and E154P; (n) K156 is substituted with K156N; (o) V167 is substituted by V167I; or (p) The substitution of E168 is E168N.

6. The adenosine deaminase according to claim 4 or 5, wherein the adenosine deaminase comprises an amino acid sequence selected from any one of SEQ ID NOs: 13 to 16 or an amino acid sequence having at least about 80% sequence identity thereto.

7. An adenosine deaminase having an amino acid substitution at one, more or all of the following positions relative to the sequence shown in SEQ ID NO: 17: W22, Q35, P47, Y50, L83, A105, D107, E108, T110, D118, G121, H122, S145, F148, R151, E154, K155 and K156, and having an amino acid sequence with about 70% to about 99.5% sequence identity with the sequence shown in SEQ ID NO:

17.

8. Adenosine deaminase according to claim 7, wherein: (a) the substitution of W22 is selected from the group consisting of: W22R, W22K and W22H; (b) the substitution of Q35 is selected from the group consisting of: Q35L, Q35V, Q35I and Q35P; (c) P47 is substituted with P47A; (d) the substitution of Y50 is selected from the group consisting of: Y50L, Y50V, Y50I and Y50P; (e) the substitution of L83 is selected from the group consisting of: L83F, L83W and L83Y; (f) A105 is substituted with a member selected from the group consisting of A105V, A105I, A105L, and A105P; (g) D107 is substituted with D107N; (h) E108 is substituted with E108S; (i) a substitution of T110 is selected from the group consisting of: T110R, T110K and T110H; (j) substitution position D118N of D118; (k) G121 is substituted with G121N; (l) H122 is substituted with H122Y; (m) S145 is substituted with S145C; (n) F148 is substituted with F148Y; (o) R151 is substituted with R151P; (p) a substitution of E154 selected from the group consisting of: E154V, E154I, E154L and E154P; (q) a substitution of K155 selected from the group consisting of: K155F, K155W and K155Y; (r) Substitution of K156 to K156N.

9. The adenosine deaminase according to claim 7 or 8, wherein the adenosine deaminase comprises an amino acid sequence selected from any one of SEQ ID NOs: 18 to 21 or an amino acid sequence having at least about 80% sequence identity thereto.

10. A base editor comprising the adenosine deaminase according to any one of claims 1 to 9.

11. The base editor of claim 10, further comprising a cytidine deaminase domain.

12. The base editor according to claim 10, further comprising a second adenosine deaminase domain, wherein the second adenosine deaminase domain is the same as or different from the adenosine deaminase according to any one of claims 1 to 9.

13. The base editor according to any one of claims 10 to 12, further comprising one or more nuclear localization sequences.

14. The base editor according to any one of claims 10 to 13, wherein the base editor further comprises a programmable DNA binding domain, and the programmable DNA binding domain is selected from a Cas9 domain or a Cas12 domain; preferably a dCas9 domain or an nCas9 domain.

15. A base editor according to any one of claims 10 to 14, wherein the base editor comprises an amino acid sequence selected from SEQ ID NOs: 46 to 54 or an amino acid sequence having at least 80% sequence identity with an amino acid sequence of SEQ ID NOs: 46 to 54.

16. A complex comprising the base editor of claim 14 or 15 and a guide RNA, wherein the guide RNA is connected to the programmable DNA binding domain of the base editor.

17. A polynucleotide encoding the adenosine deaminase according to any one of claims 1 to 9 or the base editor according to any one of claims 10 to 15.

18. A vector comprising the polynucleotide of claim 17.

19. A cell comprising the vector of claim 18.

20. A pharmaceutical composition comprising the base editor of any one of claims 10 to 15, the complex of claim 16, the vector of claim 18, or the cell of claim 19.

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