# US 11,999,947 B2 · Adenosine nucleobase editors and uses thereof

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- Number: US 11,999,947 B2
- Document ID: US11999947B2
- Status: Granted
- Kind: B2
- Application number: 18/174,569
- Filed: 2023-02-24
- Published: 2024-03-07
- Granted: 2024-06-04
- Earliest claimed: 2016-08-03
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: David R. Liu; Nicole Gaudelli
- Web page: https://patentgrep.com/patent/US11999947B2

## CPC

- C12N 15/1024: {In vivo mutagenesis using high mutation rate "mutator" host strains by inserting genetic material, e.g. encoding an error prone polymerase, disrupting a gene for mismatch repair}
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12Y 305/04004: Adenosine deaminase (3.5.4.4)
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The disclosure provides adenosine deaminases that are capable of deaminating adenosine in DNA. The disclosure also provides fusion proteins comprising a Cas9 (e.g., a Cas9 nickase) domain and adenosine deaminases that deaminate adenosine in DNA. In some embodiments, the fusion proteins further comprise a nuclear localization sequence (NLS), and/or an inhibitor of base repair, such as, a nuclease dead inosine specific nuclease (dISN).

## Claims

### Claim 1

An adenosine deaminase comprising an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, with the exception of one or more of the following substitutions H36L, P48A, L84F, A106V, D108N, S146C, S146R, R152P, E155V, I156F, and K157N in the amino acid sequence of SEQ ID NO: 1, and wherein said adenosine deaminase deaminates adenine in deoxyribonucleic acid (DNA).

### Claim 2 (depends on 1)

The adenosine deaminase of claim 1, wherein said adenosine deaminase comprises an amino acid sequence having 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.

### Claim 3

An adenosine deaminase comprising an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, with the exception of the following amino acid substitutions H36L, P48A, L84F, A106V, D108N, S146C or S146R, R152P, E155V, I156F, and K157N in the amino acid sequence of SEQ ID NO: 1, and wherein said adenosine deaminase deaminates adenine in deoxyribonucleic acid (DNA).

### Claim 4

An adenosine deaminase comprising an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, with the exception of the following amino acid substitutions H36L, P48A, L84F, A106V, D108N, S146C or S146R, R152P, E155V, I156F, and K157N, and one or more substitutions at positions selected from the group consisting of amino acid residues corresponding to positions 123, 147, 154, and 166 in the amino acid sequence of SEQ ID NO: 1, wherein said adenosine deaminase variant deaminates adenine in deoxyribonucleic acid (DNA).

### Claim 5 (depends on 1)

The adenosine deaminase of claim 1, wherein said one or more substitutions comprise the following group of substitutions: H36L, P48A, L84F, A106V, D108N, S146C or S146R, R152P, E155V, I156F, and K157N in the amino acid sequence of SEQ ID NO: 1.

### Claim 6 (depends on 3)

The adenosine deaminase of claim 3, wherein the adenosine deaminase further comprises one or more substitutions at positions selected from the group consisting of amino acid residues corresponding to positions 23, 25, 51, 70, 72, 94, 138, 147, and 154 in the amino acid sequence of SEQ ID NO: 1.

### Claim 7 (depends on 3)

The adenosine deaminase of claim 3, wherein the adenosine deaminase further comprises one or more substitutions at positions selected from the group consisting of amino acid residues corresponding to positions 123, 147, 154, and 166 of the amino acid sequence of SEQ ID NO: 1.

### Claim 8 (depends on 3)

The adenosine deaminase of claim 3, wherein the adenosine deaminase further comprises the substitution Q154R in the amino acid sequence of SEQ ID NO: 1.

### Claim 9 (depends on 1)

A base editor comprising:
a nucleic acid programmable DNA binding protein (napDNAbp) domain, wherein said napDNAbp domain when in conjunction with a bound nucleic acid, site specifically binds a nucleic acid sequence; and
an adenosine deaminase domain comprising the adenosine deaminase of claim 1.

### Claim 10 (depends on 9)

The base editor of claim 9, wherein said napDNAbp domain comprises a dead Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease active Cas9 domain.

### Claim 11 (depends on 10)

The base editor of claim 10, wherein said napDNAbp domain comprises a nCas9 domain.

### Claim 12 (depends on 9)

The base editor of claim 9, wherein said napDNAbp domain is capable of binding a nucleotide sequence that does not contain a canonical PAM sequence.

### Claim 13 (depends on 9)

The base editor of claim 9 further comprising a second adenosine deaminase domain.

### Claim 14 (depends on 13)

The base editor of claim 13, wherein the second adenosine deaminase domain comprises a second adenosine deaminase that is a TadA adenosine deaminase.

### Claim 15 (depends on 14)

The base editor of claim 14, wherein the second adenosine deaminase comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 1.

### Claim 16 (depends on 1)

A method for editing a nucleobase of a DNA sequence, the method comprising:
contacting said DNA sequence with the adenosine deaminase of claim 1; whereby a first nucleobase of said DNA sequence is converted to a second nucleobase.

### Claim 17 (depends on 16)

The method of claim 16, wherein said first nucleobase is adenine.

### Claim 18 (depends on 16)

The method of claim 16, wherein said second nucleobase is inosine.

### Claim 19 (depends on 16)

The method of claim 16, whereby a third nucleobase complementary to said first nucleobase is replaced by a fourth nucleobase complementary to said second nucleobase.

### Claim 20 (depends on 19)

The method of claim 19 whereby a second nucleobase is replaced with a fifth nucleobase that is complementary to said fourth nucleobase.

### Claim 21 (depends on 20)

The method of claim 20, wherein said fifth nucleobase is guanine.