# US 2020/0399619 A1 · Editing of ccr5 receptor gene to protect against hiv infection

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- Number: US 2020/0399619 A1
- Document ID: US20200399619A1
- Status: Published application
- Kind: A1
- Application number: 16/926,436
- Filed: 2020-07-10
- Published: 2020-12-24
- Earliest claimed: 2016-12-23
- Assignees: President and Fellows of Harvard College
- Applicants: President and Fellows of Harvard College
- Inventors: Juan Pablo Maianti; David R. Liu
- Web page: https://patentgrep.com/patent/US20200399619A1

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- A61P 31/18: for HIV
- C07K 14/7158: {for chemokines}
- C12N 9/78: acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/1138: {against receptors or cell surface proteins}
- C12Y 305/04001: Cytosine deaminase (3.5.4.1)
- C07K 2319/80: containing a DNA binding domain, e.g. Lacl or Tet-repressor
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2740/16062: by genetic engineering
- C12Y 301/21: Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
- 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
- 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)
- C12Y: ENZYMES

## Abstract

Provided herein are systems, compositions, and methods of introducing protective and/or loss-of-function variants of CCR5 and CCR2. Variants may be introduced using a CRISPR/Cas9-based nucleobase editor or other guide nucleotide sequence-programmable DNA binding protein domain-based fusion protein described herein. Further provided herein are compositions and methods of preventing and treating conditions related to HIV infection and progression as well as to AIDS.

## Claims

### Claim at position 1

1-68. (canceled)

### Claim 69

A method of editing a polynucleotide encoding a C-C chemokine receptor type 2 (CCR2) protein, the method comprising contacting the CCR2-encoding polynucleotide with:
(i) a fusion protein comprising: (a) a guide nucleotide sequence-programmable DNA binding protein domain; and (b) a cytosine deaminase domain; and
(ii) a guide nucleotide sequence targeting the fusion protein of (i) to a target cytosine (C) base in the CCR2-encoding polynucleotide,
wherein the contacting results in the deamination of the target C base by the fusion protein, resulting in a cytosine-guanine (C:G) to thymine-adenine pair (T:A) change in the CCR2-encoding polynucleotide.

### Claim 70 (depends on 69)

The method of claim 69, wherein the C to T change is in the coding sequence of the CCR2-encoding polynucleotide.

### Claim 71 (depends on 69, 70)

The method of claim 69 or 70, wherein the C to T change leads to leads to a mutation in the CCR2 protein.

### Claim 72 (depends on 71)

The method of claim 71, wherein the mutation in the CCR2 protein is a loss-of-function mutation.

### Claim 73

(canceled)

### Claim 74 (depends on 69)

The method of claim 69, wherein the method is carried out in vitro.

### Claim 75

(canceled)

### Claim 76 (depends on 69)

The method of claim 69, wherein the method is carried out in vivo.

### Claim 77 (depends on 69)

The method of claim 69, wherein the method is carried out ex vivo.

### Claim 78 (depends on 76)

The method of claim 76, wherein the method is carried out in a mammal.

### Claim 79

(canceled)

### Claim 80 (depends on 78)

The method of claim 78, wherein the mammal is human.

### Claim 81

(canceled)

### Claim 82 (depends on 69)

The method of claim 69, wherein the guide nucleotide sequence-programmable DNA binding protein domain is selected from the group consisting of: a nuclease inactive Cas9 (dCas9) domain, a nuclease inactive Cpf1 domain, a nuclease inactive Argonaute domain, and variants and combinations thereof.

### Claim 83 (depends on 69)

The method of claim 69, wherein the guide nucleotide sequence-programmable DNA-binding protein domain comprises a nuclease inactive Cas9 (dCas9) domain.

### Claim at position 17

84-90. (canceled)

### Claim 91 (depends on 69)

The method of claim 69, wherein the cytosine deaminase domain comprises a cytosine deaminase that is selected from the group consisting of APOBEC1, APOBEC2, APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, activation-induced deaminase (AID), and pmCDA1.

### Claim 92

(canceled)

### Claim 93 (depends on 69)

The method of claim 69, wherein the fusion protein of (a) further comprises a uracil glycosylase inhibitor (UGI) domain.

### Claim 94

(canceled)

### Claim 95 (depends on 93)

The method of claim 93, wherein the UGI domain is fused to the C-terminus of the guide nucleotide sequence-programmable DNA-binding protein domain.

### Claim at position 23

96-104. (canceled)

### Claim 105

A composition comprising:
(i) a fusion protein comprising: (a) a guide nucleotide sequence-programmable DNA binding protein domain; and (b) a cytosine deaminase domain; and
(ii) a guide nucleotide sequence targeting the fusion protein of (i) to a polynucleotide encoding a C-C chemokine receptor type two (CCR2) protein.

### Claim 106

A composition comprising:
(i) a fusion protein comprising: (a) a guide nucleotide sequence-programmable DNA binding protein domain; and (b) a cytosine deaminase domain;
(ii) a guide nucleotide sequence targeting the fusion protein of (i) to a polynucleotide encoding a C-C chemokine receptor type five (CCR5) protein; and
(iii) a guide nucleotide sequence targeting the fusion protein of (i) to a polynucleotide encoding a C-C chemokine receptor type 2 (CCR2) protein.

### Claim at position 26

107-114. (canceled)

### Claim 115 (depends on 105)

A method of reducing viral infection in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 105.

### Claim at position 28

116-117. (canceled)

### Claim 118 (depends on 105)

A method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 105, wherein the condition is human immunodeficiency virus (HIV) infection, re-infection, or activation from latency, acquired immune deficiency syndrome (AIDS), an immunologic disease, or a combination thereof.

### Claim at position 30

119-121. (canceled)

### Claim 122 (depends on 105)

A method of preventing a condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of the composition of claim 105, wherein the condition is human immunodeficiency virus (HIV) infection, re-infection, or activation from latency, acquired immune deficiency syndrome (AIDS), an immunologic disease, or a combination thereof.

### Claim at position 32

123-125. (canceled)

### Claim 126 (depends on 105)

A kit comprising the composition of claim 105.