# US 8,772,025 B2 · Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression

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- Number: US 8,772,025 B2
- Document ID: US8772025B2
- Status: Granted
- Kind: B2
- Application number: 12/931,265
- Filed: 2011-01-27
- Published: 2011-06-30
- Granted: 2014-07-08
- Earliest claimed: 2007-07-12
- Assignees: Sangamo BioSciences, Inc.
- Applicants: Trevor Collingwood; Gregory J. Cost
- Inventors: Trevor Collingwood; Gregory J. Cost
- Web page: https://patentgrep.com/patent/US8772025B2

## CPC

- C12N 15/907: {in mammalian cells}
- C07K 14/4703: {Inhibitors; Suppressors}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 9/1051: {Hexosyltransferases (2.4.1)}
- C12N 15/85: for animal cells
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Disclosed herein are methods and compositions for inactivating a FUT8 gene, using fusion proteins comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.

## Claims

### Claim 1

A zinc finger 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, F2, F3, and F4 comprise the following amino acid sequences:
F1: QSSDLSR (SEQ ID NO: 9);
F2: TSGNLTR (SEQ ID NO: 10);
F3: RSDDLSK (SEQ ID NO: 11); and
F4: DRSALAR (SEQ ID NO: 12).

### Claim 2 (depends on 1)

An isolated polynucleotide encoding the zinc finger DNA-binding domain of claim 1.

### Claim 3 (depends on 1)

A fusion protein comprising a zinc finger DNA-binding domain according to claim 1 and at least one cleavage domain or at least one cleavage half-domain.

### Claim 4 (depends on 3)

The fusion protein of claim 3, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

### Claim 5 (depends on 4)

The fusion protein of claim 4, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

### Claim 6 (depends on 3)

A polynucleotide encoding the fusion protein according to claim 3.

### Claim 7 (depends on 3)

An isolated cell comprising the fusion protein according to claim 3.

### Claim 8 (depends on 7)

The cell of claim 7, wherein Fut8 is partially inactivated.

### Claim 9 (depends on 7)

The cell of claim 7, wherein Fut8 is fully inactivated.

### Claim 10 (depends on 7)

The cell of claim 7, wherein one or more additional genes are partially or fully inactivated.

### Claim 11 (depends on 10)

The cell of claim 10, wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.

### Claim 12

A zinc finger DNA-binding domain, wherein the DNA-binding domain comprises five zinc finger recognition regions ordered F1 to F5 from N-terminus to C-terminus, and wherein F1, F2, F3, F4, and F5 comprise the following amino acid sequences:
FI: RSDNLRE (SEQ ID NO: 26);
F2: NNTQLIE (SEQ ID NO: 27);
F3: TSSILSR (SEQ ID NO: 28);
F4 RSDNLSA (SEQ ID NO: 29); and
F5: RKDTRIT (SEQ ID NO: 30).

### Claim 13 (depends on 12)

An isolated polynucleotide encoding the zinc finger DNA-binding domain of claim 12.

### Claim 14 (depends on 12)

A fusion protein comprising a zinc finger DNA-binding domain according to claim 12 and at least one cleavage domain or at least one cleavage half-domain.

### Claim 15 (depends on 14)

The fusion protein of claim 14, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

### Claim 16 (depends on 14)

The fusion protein of claim 14, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

### Claim 17 (depends on 14)

A polynucleotide encoding the fusion protein according to claim 14.

### Claim 18 (depends on 14)

An isolated cell comprising the protein according to claim 14.

### Claim 19 (depends on 18)

The cell of claim 18, wherein Fut8 is partially inactivated.

### Claim 20 (depends on 18)

The cell of claim 18, wherein Fut8 is fully inactivated.

### Claim 21 (depends on 18)

The cell of claim 18, wherein one or more additional genes are partially or fully inactivated.

### Claim 22 (depends on 21)

The cell of claim 21, wherein dihydrofolate reductase (DHFR) and glutamine synthetase (GS) have been inactivated.