# US 7,951,925 B2 · Methods and compositions for gene inactivation

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- Number: US 7,951,925 B2
- Document ID: US7951925B2
- Status: Granted
- Kind: B2
- Application number: 11/805,707
- Filed: 2007-05-23
- Published: 2008-07-03
- Granted: 2011-05-31
- Earliest claimed: 2006-05-25
- Assignees: Sangamo BioSciences, Inc.
- Inventors: Dale Ando; Michael C. Holmes; Gary Ka Leong Lee
- Web page: https://patentgrep.com/patent/US7951925B2

## CPC

- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/46: Viral antigens
- A61P 37/00: Drugs for immunological or allergic disorders
- C07K 14/47: from mammals
- C07K 14/4703: {Inhibitors; Suppressors}
- C12N 5/10: Cells modified by introduction of foreign genetic material
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61P 31/18: for HIV
- C12N 15/86: Viral vectors
- C12N 2710/10343: viral genome or elements thereof as genetic vector
- C12N 2710/10345: Special targeting system for viral vectors
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- 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)

## Abstract

Disclosed herein are methods and compositions for inactivating CCR-5 genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs, such as adenovirus (Ad) vectors, and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided.

## Claims

### Claim 1

A protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F3, and F4 comprise the following amino acid sequences:
F1: DRSNLSR
(SEQ ID NO: 2)
F3: RSDNLAR
(SEQ ID NO: 4)
F4: TSGNLTR.
(SEQ ID NO: 8)

### Claim 2 (depends on 1)

The protein according to claim 1, wherein F2 comprises the amino acid sequence ISSNLNS (SEQ ID NO:5).

### Claim 3 (depends on 1)

The protein according to claim 1, wherein F2 comprises the amino acid sequence VSSNLTS (SEQ ID NO:6).

### Claim 4 (depends on 1)

A protein according to claim 1, further comprising a cleavage domain.

### Claim 5 (depends on 4)

The protein of claim 4, wherein the cleavage domain is a cleavage half-domain.

### Claim 6 (depends on 5)

The protein of claim 5, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

### Claim 7 (depends on 5)

The protein of claim 5, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

### Claim 8 (depends on 1)

A polynucleotide encoding the protein of claim 1.

### Claim 9 (depends on 8)

A gene delivery vector comprising a polynucleotide according to claim 8.

### Claim 10 (depends on 9)

The gene delivery vector of claim 9, wherein the vector is an adenovirus vector.

### Claim 11 (depends on 8)

The gene delivery vector of claim 8, wherein the adenovirus vector is an Ad5/35 vector.

### Claim 12 (depends on 1)

An isolated cell comprising the protein of claim 1.

### Claim 13 (depends on 8)

An isolated cell comprising the polynucleotide of claim 8.

### Claim 14 (depends on 12)

The cell of claim 12, wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.

### Claim 15 (depends on 14)

The cell of claim 14, wherein the T-cell is a CD4+ cell.

### Claim 16 (depends on 13)

The cell of claim 13, wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.

### Claim 17 (depends on 16)

The cell of claim 16, wherein the T-cell is a CD4+ cell.

### Claim 18 (depends on 8)

A method for inactivating the CCR-5 gene in a human cell, the method comprising administering to the cell a polynucleotide according to claim 8.

### Claim 19 (depends on 18)

The method of claim 18, wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.

### Claim 20 (depends on 19)

The method of claim 19, wherein the T-cell is a CD4+ cell.

### Claim 21 (depends on 12)

The isolated cell of claim 12 further comprising a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition helices as follows:
F1: RSDNLSV
(SEQ ID NO: 10)
F2: QKINLQV
(SEQ ID NO: 17)
F3: RSDVLSE
(SEQ ID NO: 12)
F4: QRNHRTT.
(SEQ ID NO: 13)

### Claim 22 (depends on 13)

The isolated cell of claim 13, further comprising a polynucleotide encoding a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition helices as follows:
F1: RSDNLSV (SEQ ID NO: 10)
F2: QKINLQV (SEQ ID NO: 17)
F3: RSDVLSE (SEQ ID NO: 12)
F4: QRNHRTT (SEQ ID NO: 13).