Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases

• US 8,956,828 B2
• Filed: 11/10/2010
• Issued: 02/17/2015
• Est. Priority Date: 11/10/2009
• Status: Active Grant

• Current Assignee: Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Therapeutics, Inc.
• Original Assignee: Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Biosciences, Inc.
• Inventors: Genovese, Pietro, Gregory, Philip D., Holmes, Michael C., Naldini, Luigi, Paschon, David, Zhang, Lei, Bonini, Maria Chiara, Provasi, Elena
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/927,292
• Publication Number:
  US 20110158957A1
• Time in Patent Office: 1,560 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: A61K 35/00 Medicinal preparations containing materials or reaction products thereof with undetermined constitution
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: 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
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C07K 2319/95 containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for inactivating TCR genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided. Disclosed herein are also methods and compositions for expressing a functional exogenous TCR in the absence of endogenous TCR expression in T lymphocytes, including lymphocytes with a central memory phenotype. Polynucleotides encoding exogenous TCR, vectors comprising polynucleotides encoding exogenous TCR and cells comprising polynucleotides encoding exogenous TCR and/or cells comprising exogenous TCR are also provided.

Claims

1. An isolated zinc finger nuclease (ZFN) comprising

a zinc finger protein that binds to a target site in a T-cell receptor (TCR) gene, wherein the zinc finger protein comprises four, five or six zinc finger domains ordered F1 to F4 for four-finger proteins, F1 to F5 for five-finger proteins and F1 to F6 for six-finger proteins, wherein the zinc finger protein is selected from the group consisting of a protein comprising the following amino acid sequences in the recognition regions:

(i) F1;

QSGDLTR (SEQ ID NO;

2);

F2;

QRTHLKA (SEQ ID NO;

3);

F3;

QSGDRNK (SEQ ID NO;

     4);

F4;

DRSNLSR (SEQ ID NO;

5); and

F5;

RSDALTQ (SEQ ID NO;

6);

(ii) F1;

TSGSLSR (SEQ ID NO;

8);

F2;

QSSVRNS (SEQ ID NO;

9);

F3;

RSDNLST (SEQ ID NO;

10);

F4;

DRSALAR (SEQ ID NO;

11); and

F5;

LKQNLDA (SEQ ID NO;

12);

(iii) F1;

DRSALSR (SEQ ID NO;

14);

F2;

QSGHLSR (SEQ ID NO;

15);

F3;

DRSDLSR (SEQ ID NO;

16);

F4;

RSDALSR (SEQ ID NO;

17); and

F5;

DRSDLSR (SEQ ID NO;

16);

(iv) F1;

DRSNLSR (SEQ ID NO;

5);

F2;

QKTSLQA (SEQ ID NO;

19);

F3;

DRSALSR (SEQ ID NO;

14);

F4;

QSGNLAR (SEQ ID NO;

20);

F5;

GKEELNE (SEQ ID NO;

21); and

F6;

RSSDLSR (SEQ ID NO;

22);

(v) F1;

GNVDLIE (SEQ ID NO;

24);

F2;

RSSNLSR (SEQ ID NO;

25);

F3;

RSDALSV (SEQ ID NO;

26);

F4;

DSSHRTR (SEQ ID NO;

27); and

F5;

WRSCRSA (SEQ ID NO;

28);

(vi) F1;

DSSDRKK (SEQ ID NO;

30);

F2;

RSDNLSV (SEQ ID NO;

31);

F3;

RRFILRG (SEQ ID NO;

32);

F4;

QSGDLTR (SEQ ID NO;

2); and

F5;

TSGSLTR (SEQ ID NO;

33);

(vii) F1;

QSGDLTR (SEQ ID NO;

2);

F2;

QTSTLSK (SEQ ID NO;

35);

F3;

QSGHLSR (SEQ ID NO;

15);

F4;

DRSDLSR (SEQ ID NO;

16); and

F5;

RSDALAR (SEQ ID NO;

36);

(viii) F1;

QSGDLTR (SEQ ID NO;

2);

F2;

WRSSLAS (SEQ ID NO;

38);

F3;

QSGDLTR (SEQ ID NO;

2);

F4;

HKWVLRQ (SEQ ID NO;

39); and

F5;

DRSNLTR (SEQ ID NO;

40);

(ix) F1;

QSGDLTR (SEQ ID NO;

2);

F2;

QWGTRYR (SEQ ID NO;

42);

F3;

ERGTLAR (SEQ ID NO;

43);

F4;

RSDNLRE (SEQ ID NO;

44);

F5;

QSGDLTR (SEQ ID NO;

2); and

F6;

TSGSLTR (SEQ ID NO;

33);

(x) F1;

RSAHLSR (SEQ ID NO;

46);

F2;

DRSDLSR (SEQ ID NO;

16);

F3;

RSDHLSV (SEQ ID NO;

47); and

F4;

QNNHRIT (SEQ ID NO;

48);

(xi) F1;

QRSNLVR (SEQ ID NO;

50);

F2;

RNDDRKK (SEQ ID NO;

51);

F3;

TSGNLTR (SEQ ID NO;

52); and

F4;

TSANLSR (SEQ ID NO;

53);

(xii) F1;

DRSTLRQ (SEQ ID NO;

55);

F2;

QRSNLVR (SEQ ID NO;

50);

F3;

RNDDRKK (SEQ ID NO;

51);

F4;

RSAHLSR (SEQ ID NO;

46); and

F5;

QSGHLSR (SEQ ID NO;

15);

(xiii) F1;

QRSNLVR (SEQ ID NO;

50);

F2;

RNDDRKK (SEQ ID NO;

51);

F3;

QSGHLAR (SEQ ID NO;

56); and

F4;

QSGHLSR (SEQ ID NO;

15);

(xiv) F1;

RSAHLSR (SEQ ID NO;

46);

F2;

DRSDLSR (SEQ ID NO;

16);

F3;

RSDHLSV (SEQ ID NO;

47); and

F4;

QNNHRIT (SEQ ID NO;

48);

(xv) F1;

RSDVLSA (SEQ ID NO;

59);

F2;

DRSNRIK (SEQ ID NO;

60);

F3;

RSDVLSE (SEQ ID NO;

61);

F4;

QSGNLAR (SEQ ID NO;

20); and

F5;

QSGSLTR (SEQ ID NO;

62);

(xvi) F1;

RSDHLST (SEQ ID NO;

64);

F2;

RSDNLTR (SEQ ID NO;

65);

F3;

DRSNLSR (SEQ ID NO;

5);

F4;

TSSNRKT (SEQ ID NO;

66);

F5;

RSANLAR (SEQ ID NO;

67);

F6;

RNDDRKK (SEQ ID NO;

51);

(xvii) F1;

RSDHLST (SEQ ID NO;

64);

F2;

RSDNLTR (SEQ ID NO;

65);

F3;

DRSNLSR (SEQ ID NO;

5);

F4;

LQFNRNQ (SEQ ID NO;

68);

F5;

RSANLAR (SEQ ID NO;

67); and

F6;

RNDDRKK (SEQ ID NO;

51);

(xviii) F1;

RSDHLST (SEQ ID NO;

64);

F2;

RSDNLTR (SEQ ID NO;

65);

F3;

DSSNLSR (SEQ ID NO;

69);

F4;

LRFNLSN (SEQ ID NO;

70);

F5;

RSANLAR (SEQ ID NO;

67); and

F6;

RNDDRKK (SEQ ID NO;

51);

(xix) F1;

RSDHLST (SEQ ID NO;

64);

F2;

RSDNLTR (SEQ ID NO;

65);

F3;

DSSNLSR (SEQ ID NO;

69);

F4;

LHFQLTG (SEQ ID NO;

71);

F5;

RSANLAR (SEQ ID NO;

67); and

F6;

RNDDRKK (SEQ ID NO;

51);

(xx) F1;

RSDVLSA (SEQ ID NO;

59);

F2;

DRSNRIK (SEQ ID NO;

60);

F3;

RSDVLSE (SEQ ID NO;

61);

F4;

QSGNLAR (SEQ ID NO;

20); and

F5;

QSGSLTR (SEQ ID NO;

62);

(xxi) F1;

DRSNLSR (SEQ ID NO;

5);

F2;

LKFALAN (SEQ ID NO;

72);

F3;

RSDVLSE (SEQ ID NO;

61);

F4;

QSGNLAR (SEQ ID NO;

20); and

F5;

QSGSLTR (SEQ ID NO;

62);

(xxii) F1;

RSDVLSA (SEQ ID NO;

59);

F2;

DRSNRIK (SEQ ID NO;

60);

F3;

RSDVLSE (SEQ ID NO;

61);

F4;

QSGNLAR (SEQ ID NO;

20); and

F5;

QSGALAR (SEQ ID NO;

73); and

(xxiii) F1;

RLSVLTI (SEQ ID NO;

74);

F2;

DRANLTR (SEQ ID NO;

75);

F3;

RSDVLSE (SEQ ID NO;

61);

F4;

QSGNLAR (SEQ ID NO;

20); and

F5;

QSGALAR (SEQ ID NO;

73);

and a cleavage domain.

2. The zinc finger nuclease of claim 1, wherein the TCR gene is a human TCR α

gene or human TCR β

gene.

3. The zinc finger nuclease of claim 1, wherein the cleavage domain comprises a wild-type or engineered FokI cleavage half-domain.

4. A polynucleotide encoding a zinc finger nuclease according to claim 1.

5. A gene delivery vector comprising the polynucleotide of claim 4.

6. The gene delivery vector of claim 5, wherein the vector is an adenoviral or lentiviral vector.

7. The gene delivery vector of claim 6, wherein the adenoviral vector is an AdF5/F35 vector or the lentiviral vector is an integrase-defective lentiviral vector (IDLV).

8. An isolated cell or a cell line comprising a zinc finger nuclease according to claim 1.

9. An isolated cell or a cell line comprising a polynucleotide according to claim 4.

10. An isolated cell in which a TCR gene has been inactivated, wherein the cell is descended from the isolated cell or cell line of claim 8.

11. A method of inactivating a TCR gene in an isolated cell, the method comprising expressing a zinc finger nuclease according to claim 1 in the isolated cell.

12. The method of claim 11, further comprising

introducing into the isolated cell a sequence encoding one or more TCR genes specific for an MHC/antigen complex or a chimeric antigen receptor.

13. The method of claim 12, wherein the sequence is integrated at or near the cleavage site.

14. The method of claim 12, wherein the sequence is integrated into a safe-harbor locus.

15. The method of claim 11, wherein the isolated cell is selected from the group consisting of a stem cell, a progenitor cell, and a T-cell.