# US 2023/0272425 A1 · Methods and compositions for evolving base editors using phage-assisted continuous evolution (pace)

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- Number: US 2023/0272425 A1
- Document ID: US20230272425A1
- Status: Published application
- Kind: A1
- Application number: 18/178,048
- Filed: 2023-03-03
- Published: 2023-08-31
- Earliest claimed: 2017-07-28
- Assignees: President and Fellows of Harvard College; The Broad Institute, Inc.
- Applicants: President and Fellows of Harvard College; The Broad Institute, Inc.
- Inventors: David R. Liu; Benjamin Thuronyi; Christopher Gerard Wilson
- Web page: https://patentgrep.com/patent/US20230272425A1

## CPC

- C07K 14/32: from Bacillus (G)
- C12N 15/86: Viral vectors
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/224: {Class 1 CAS enzyme complex, e.g. multi-enzyme}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/2497: {hydrolysing N- glycosyl compounds (3.2.2)}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12Y 305/04005: Cytidine deaminase (3.5.4.5)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

The instant specification provides for evolved base editors which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system. In particular, the instant specification provides for evolved cytidine base editors (e.g., based on APOBEC1, CDA, or AID cytidine deaminase domains) which overcome deficiencies of those in art (including increased efficiency and/or decreased requirement for specific sequence-context at an editing site) and which are obtained a result of a phage-assisted continuous evolution (PACE) system.

## Claims

### Claim 1

A cytidine deaminase comprising an amino acid sequence that is at least 80% identical to amino acid residues 2-162 of SEQ ID consisting of H102X1, D104X2, and V115X3 relative to SEQ ID NO: 1, or a corresponding mutation(s) in another cytidine deaminase, wherein X1 is any amino acid other than H, X2 is any amino acid other than D, and X3 is any amino acid other than V.

### Claim 2 (depends on 1)

The cytidine deaminase of claim 1 comprising amino acid residues 2-162 of SEQ ID NO: 1, wherein the cytidine deaminase comprises one or more mutations selected from the group consisting of H102X1, D104X2, and V115X3 relative to SEQ ID NO: 1, wherein X1 is any amino acid other than H, X2 is any amino acid other than D, and X3 is any amino acid other than V.

### Claim at position 3

3-7. (canceled)

### Claim 8 (depends on 1)

The cytidine deaminase of claim 1, wherein the cytidine deaminase comprises amino acid residues 2-162 of SEQ ID NO: 5.

### Claim at position 5

9-29. (canceled)

### Claim 30

A cytidine deaminase comprising an amino acid sequence that is at least 80% identical to amino acid residues 2-208 of SEQ ID NO: 3, wherein the cytidine deaminase comprises one or more mutations selected from the group consisting of H10X1, F23X2, V75X3, K120X4, A123X5, C158X6, I193X7, I195X8, and V197X9 relative to SEQ ID NO: 3, or a corresponding mutation(s) in another cytidine deaminase, wherein X1, is any amino acid other than H, X2 is any amino acid other than F, X3 and X9 are any amino acid other than V, X4 is any amino acid other than K, X5 is any amino acid other than A, X6 is any amino acid other than C, and X7 and X8 are any amino acid other than I.

### Claim 31 (depends on 30)

The cytidine deaminase of claim 30 comprising amino acid residues 2-208 of SEQ ID NO: 3, wherein the cytidine deaminase comprises one or more mutations selected from the group consisting of H10X1, F23X2, V75X3, K120X4, A123X5, C158X6, I193X7, I195X8, and V197X9 relative to SEQ ID NO: 3, wherein X1, is any amino acid other than H, X2 is any amino acid other than F, X3 and X9 are any amino acid other than V, X4 is any amino acid other than K, X5 is any amino acid other than A, X6 is any amino acid other than C, and X7 and X8 are any amino acid other than I.

### Claim at position 8

32-44. (canceled)

### Claim 45 (depends on 30)

The cytidine deaminase of claim 30, wherein the cytidine deaminase comprises amino acid residues 2-208 of SEQ ID NO: 7.

### Claim at position 10

46-47. (canceled)

### Claim 48

A cytidine deaminase comprising an amino acid sequence that is at least 80% identical to amino acid residues 3-229 of SEQ ID NO: 4, wherein the cytidine deaminase comprises one or more mutations selected from the group consisting of E4X1, H122X2, D124X3, R154X4, A165X5, P201X6, and F205X7 relative to SEQ ID NO: 4, or a corresponding mutation(s) in another cytidine deaminase, wherein X1 is any amino acid other than E, X2 is any amino acid other than H, X3 is any amino acid other than D, X4 is any amino acid other than R, X5 is any amino acid other than A, X6 is any amino acid other than P, and X7 is any amino acid other than F.

### Claim 49 (depends on 48)

The cytidine deaminase of claim 48 comprising amino acid residues 3-229 of SEQ ID NO: 4, wherein the cytidine deaminase comprises one or more mutations selected from the group consisting of E4X1, H122X2, D124X3, R154X4, A165X5, P201X6, and F205X7 relative to SEQ ID NO: 4, wherein X1 is any amino acid other than E, X2 is any amino acid other than H, X3 is any amino acid other than D, X4 is any amino acid other than R, X5 is any amino acid other than A, X6 is any amino acid other than P, and X7 is any amino acid other than F.

### Claim at position 13

50-60. (canceled)

### Claim 61 (depends on 48)

The cytidine deaminase of claim 48, wherein the cytidine deaminase comprises amino acid residues 3-229 of SEQ ID NO: 8.

### Claim at position 15

62-64. (canceled)

### Claim 65 (depends on 30)

A fusion protein comprising:
(i) a nucleic acid programmable DNA binding protein (napDNAbp) and
(ii) the cytidine deaminase of claim 30.

### Claim at position 17

66-93. (canceled)

### Claim 94 (depends on 65)

A complex comprising the fusion protein of claim 65 and an RNA bound to the napDNAbp of the fusion protein.

### Claim at position 19

95-121. (canceled)

### Claim 122 (depends on 94)

A method comprising contacting a nucleic acid molecule with the complex of claim 94.

### Claim at position 21

123-152. (canceled)

### Claim 153

A fusion protein comprising a Cas9 domain, a uracil DNA glycosylase inhibitor (UGI) domain, and a C-intein, optionally wherein the fusion comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to
(SEQ ID NO: 138)
MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNC

### Claim at position 23

154-175. (canceled)

### Claim 176 (depends on 30)

A nucleic acid encoding the cytidine deaminase of claim 30.

### Claim 177 (depends on 176)

A vector comprising the nucleic acid of claim 176.

### Claim 178 (depends on 30)

A cell comprising the cytidine deaminase of claim 30.

### Claim 179 (depends on 65)

A pharmaceutical composition comprising the fusion protein of claim 65 and a pharmaceutically acceptable excipient.

### Claim 180 (depends on 65)

The fusion protein of claim 65 further comprising a uracil glycosylase inhibitor domain (UGI).

### Claim 181

A base editor comprising an amino acid sequence that is at least 80% identical to the one or more of the amino acid sequences selected from the group containing SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 20.

### Claim 182 (depends on 30, 181)

The base editor of claim 181 wherein the amino acid sequence that is at least 80% identical to SEQ ID NO: 18 and wherein the base editor comprises the cytidine deaminase of claim 30.

### Claim 183 (depends on 181)

The base editor of claim 181 wherein the amino acid sequence is the amino acid sequence of SEQ ID NO: 18.