ADENOSINE DEAMINASE BASE EDITORS AND METHODS OF USING SAME TO MODIFY A NUCLEOBASE IN A TARGET SEQUENCE

1 . A fusion protein comprising a polynucleotide programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant comprising an arginine or a threonine at amino acid position 147 of the following sequence and having at least 90% identity to the following sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST (SEQ ID NO: 4), or a fragment thereof lacking only the first methionine.

2 . The fusion protein of claim 1 , wherein the adenosine deaminase variant comprises an arginine at amino acid position 147.

3 . The fusion protein of claim 1 , wherein the adenosine deaminase variant further comprises one or more of the following alterations: I76Y, V82S, Q154S, Y123H, T166R, and Q154R.

4 . The fusion protein of claim 1 , wherein the adenosine deaminase variant further comprises one or more of the following alterations: I76Y, Y147T, and Q154S.

5 . The fusion protein of claim 1 , wherein the adenosine deaminase variant comprises the following alterations: Y123H, Y147R, and Q154R.

6 . The fusion protein of claim 1 , wherein the adenosine deaminase variant comprises the following alterations: I76Y, V82S, Y123H, Y147R, and Q154R.

7 . The fusion protein of claim 1 , wherein the adenosine deaminase variant comprises a combination of alterations selected from the following:

Y123H, Y147R, and Q154R;

Y147R and Q154R;

Y147R, Q154R, and T166R;

Y147T and Q154R;

Y147T and Q154S;

I76Y, Y123H, Y147R, and Q154R;

V82S and Y147R;

V82S, Y123H, and Y147R;

V82S; Y123H, Y147R, and Q154R;

I76Y, V82S, Y123H, Y147R, and Q154R;

Y147R and Q154S; and

V82S, Y123H, and Y147T.

8 . The fusion protein of claim 1 , wherein the fusion protein further comprises a second adenosine deaminase variant or a second adenosine deaminase.

9 . The fusion protein of claim 8 , wherein the second adenosine deaminase comprises a wild-type adenosine deaminase.

10 . The fusion protein of claim 1 , wherein the adenosine deaminase variant is TadA*8.1, TadA*8.2, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.15, TadA*8.16, TadA*8.19, TadA*8.20, TadA*8.20, TadA*8.21, TadA*8.24.

11 . The fusion protein of claim 1 , wherein the fusion protein is ABE8.1-m, ABE8.2-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.15-m, ABE8.16-m, ABE8.19-m, ABE8.20-m, ABE8.20-m, ABE8.21-m, ABE8.24-m, ABE8.1-d, ABE8.2-d, ABE8.8-d, ABE8.9-d, ABE8.10-d, ABE8.11-d, ABE8.12-d, ABE8.13-d, ABE8.15-d, ABE8.16-d, ABE8.19-d, ABE8.20-d, ABE8.20-d, ABE8.21-d, or ABE8.24-d.

12 . The fusion protein of claim 1 , wherein the adenosine deaminase variant comprises a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal or C-terminal amino acid residues relative to SEQ ID NO: 4.

13 . The fusion protein of claim 1 , wherein the polynucleotide programmable DNA binding domain comprises a Cas9 domain.

14 . The fusion protein of claim 13 , wherein the Cas9 domain is a Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), a Streptococcus pyogenes Cas9 (SpCas9), or variants thereof.

15 . The fusion protein of claim 13 , wherein the Cas9 domain is nuclease inactive variant or a nickase variant.

16 . The fusion protein of claim 1 , comprising a linker between the polynucleotide programmable DNA binding domain and the adenosine deaminase variant.

17 . The fusion protein of claim 1 , further comprising one or more nuclear localization signals.

18 . The fusion protein of claim 1 , wherein the adenosine deaminase variant has at least 95% identity to SEQ ID NO: 4.

19 . A polynucleotide encoding the fusion protein of claim 1 .

20 . An expression vector comprising the polynucleotide of claim 19 .

21 . The expression vector of claim 20 , wherein the vector is a viral vector selected from the group consisting of adeno-associated virus (AAV), retroviral vector, adenoviral vector, lentiviral vector, Sendai virus vector, and herpesvirus vector.

22 . A cell comprising the fusion protein of claim 1 .

23 . A base editor comprising the fusion protein of claim 1 in a complex with one or more guide polynucleotides.

24 . A pharmaceutical composition comprising the fusion protein of claim 1 or a polynucleotide encoding the fusion protein of claim 1 , and a pharmaceutically acceptable excipient.

25 . A kit comprising the fusion protein of claim 1 .

26 . A method for base editing comprising contacting a polynucleotide sequence with the fusion protein of claim 1 , wherein the adenosine deaminase variant deaminates a nucleobase in the polynucleotide, thereby editing the polynucleotide sequence.

27 . A method of correcting a genetic defect in a subject, the method comprising administering to the subject:

(i) a base editor comprising or consisting essentially of the fusion protein of claim 1 , or

(ii) a polynucleotide encoding said base editor and one or more guide polynucleotides that direct the base editor to deaminate a target nucleobase in a target nucleotide sequence of the subject, thereby correcting the genetic defect.

28 . The method of claim 27 , comprising delivering the base editor, or polynucleotide encoding said base editor, and one or more guide polynucleotides to a cell of the subject.

29 . The method of claim 27 , wherein the subject is a mammal or a human.

30 . The method of claim 27 , wherein the deamination of the target nucleobase replaces the target nucleobase with a wild type nucleobase.

31 . A fusion protein comprising a polynucleotide programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant comprising a histidine at position 123, an arginine at position 147, and an arginine at position 154 of the following sequence and having at least 95% identity to the following sequence: sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST (SEQ ID NO: 4), or a fragment thereof lacking only the first methionine.

32 . A fusion protein comprising a polynucleotide programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant comprising an tyrosine at position 76, a threonine at position 147, and a serine at position 154 of the following sequence and having at least 95% identity to the following sequence: sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST (SEQ ID NO: 4), or a fragment thereof lacking only the first methionine.

33 . A fusion protein comprising a polynucleotide programmable DNA binding domain and at least one base editor domain comprising an adenosine deaminase variant comprising an tyrosine at position 76, a serine at position 82, a histidine at position 123, an arginine at position 147, and an arginine at position 154 of the following sequence and having at least 95% identity to the following sequence: sequence: MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHHSRIGRVVFGVRNAKTGAAGSLMDVLHYP GMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSST (SEQ ID NO: 4), or a fragment thereof lacking only the first methionine.