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Methods and compositions for inactivating alpha 1,6 fucosyltransferase (FUT8) gene expression

  • US 9,890,395 B2
  • Filed: 03/24/2016
  • Issued: 02/13/2018
  • Est. Priority Date: 07/12/2007
  • Status: Active Grant
First Claim
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1. A method of producing an antibody in an isolated cell, the method comprising:

  • inactivating an endogenous cellular FUT8 gene in an isolated cell by introducing one or more nucleic acids encoding first and second zinc finger nucleases, each zinc finger nuclease comprising a cleavage domain and a zinc finger protein, wherein the at least one of the zinc finger nucleases comprises a zinc finger DNA-binding domain comprising four, five or six zinc finger recognition regions ordered F1 to F4, F1 to F5 or F1 to F6 from N-terminus to C-terminus, and wherein F1 to F4, F1 to F5 or F1 to F6 comprise amino acid sequences as follows;

    (i) 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);

    (ii) F1;

    RSDVLSA (SEQ ID NO;

    14);

    F2;

    QNATRIN (SEQ ID NO;

    15);

    F3;

    DRSNLSR (SEQ ID NO;

    16); and

    F4;

    RLDNRTA (SEQ ID NO;

    17);

    (iii) F1;

    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); and

    (iv) F1;

    RSDNLSV (SEQ ID NO;

    19);

    F2;

    QNATRIN (SEQ ID NO;

    15);

    F3;

    RSDNLST (SEQ ID NO;

    20) or RSDHLSQ (SEQ ID NO;

    24);

    F4;

    QSATRTK (SEQ ID NO;

    21);

    F5 RSDNLSR (SEQ ID NO;

    22); and

    F6;

    RNDNRKT (SEQ ID NO;

    23);

    wherein the first and second zinc finger nucleases cleave and inactivate the endogenous cellular FUT8 gene in the isolated cell; and

    introducing an exogenous sequence encoding an antibody into the cell, wherein the antibody is produced in the isolated cell.

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