Genome editing of a;locus using nucleases

• US 9,567,573 B2
• Filed: 04/25/2011
• Issued: 02/14/2017
• Est. Priority Date: 04/26/2010
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Gregory, Philip D., Holmes, Michael C.
• Primary Examiner(s): HOLLAND, PAUL J
• Application Number:
  US13/066,801
• Publication Number:
  US 20110265198A1
• Time in Patent Office: 2,122 Days
• Field of Search: 435/69.1, 435/366, 435/455, 435/372.2, 435/372.3, 435/456, 530/350, 536/23.4
• US Class Current: 1/1
• CPC Class Codes: A01K 2207/05 Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
• CPC Class Codes: A01K 2217/07 Animals genetically altered by homologous recombination
• CPC Class Codes: A01K 2227/105 Murine
• CPC Class Codes: A01K 2267/03 Animal model, e.g. for test or diseases
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• 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 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for genome editing of a Rosa locus, using fusion proteins comprising a DNA binding domain 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

1. An isolated fusion protein comprising a Fokl nuclease domain and one or more TAL effector (TALE) polypeptides comprising a plurality of TALE repeat domains, wherein each TALE repeat domain comprises a RVD, wherein:

(i) each RVD of said TALE repeat domains bind to a target site in a Rosa gene, wherein said target site is selected from the group consisting of the nucleotide sequence of SEQ ID NO;

28, SEQ ID NO;

29, SEQ ID NO;

79, SEQ ID NO;

80, SEQ ID NO;

81, SEQ ID NO;

82, SEQ ID NO;

87, SEQ ID NO;

88, SEQ ID NO;

89, and SEQ ID NO;

90; and

further wherein said RVD comprises the amino acids NI, HI, Al, CI, DI, El, RI or KI which recognizes adenine (A) in said target site;

the amino acids HD, AD, KD, YD, ED, AD, ND or RD which recognizes cytosine (C) in said target site;

the amino acids NN, DK, DH, KN, EN, AN, CN, GN, FN, AK, NK or CK which recognizes guanine (G) in said target site; and

the amino acids NG, KG MG, QG, RG, AA, QA, IG, IP, LA, YG, HG, SG, VG, IA or VA which recognizes thymine (T) in said target site; and

(ii) said fusion protein cleaves said Rosa gene.

2. The isolated fusion protein of claim 1, wherein said Fokl nuclease domain is naturally occurring or engineered.

3. A polynucleotide encoding said isolated fusion protein of claim 1.

4. An isolated cell comprising said isolated fusion protein of claim 1, wherein said isolated cell is not a human embryonic cell.

5. An isolated cell comprising the polynucleotide of claim 3, wherein said isolated cell is not a human embryonic cell.

6. The isolated cell of claim 4, wherein said isolated cell is a rat or mouse embryonic cell.

7. A composition comprising said isolated fusion protein of claim 1 and a pharmaceutically acceptable excipient.

8. A composition comprising said polynucleotide of claim 3 and a pharmaceutically acceptable excipient.

9. A method for cleaving one or more Rosa genes in an isolated rat or mouse cell, said method comprising:

introducing into said isolated rat or mouse cell, one or more isolated fusion proteins of claim 1 into said isolated rat or mouse cell, such that the one or more Rosa gene are cleaved.

10. The method of claim 9, wherein said one or more fusion proteins are introduced by transforming said isolated rat or mouse cell with polynucleotides encoding said isolated fusion proteins.

11. A method of introducing an exogenous polynucleotide sequence into the genome of an isolated rat or mouse cell, said method comprising:

cleaving one or more Rosa genes by the method of claim 9; and

contacting said isolated rat or mouse cell with an exogenous polynucleotide sequence;

wherein cleavage of said one or more Rosa genes stimulates integration of said exogenous polynucleotide sequence into the genome of said isolated cell by homologous recombination.

12. The method of claim 11, wherein said exogenous polynucleotide sequence is integrated into the genome via nucleic acid replication processes.

13. The method of claim 11, wherein said exogenous polynucleotide sequence is integrated into the genome via non-homology dependent targeted integration.

14. A method of modifying a Rosa gene sequence in the genome of an isolated rat or mouse cell, said method comprising:

cleaving one or more Rosa genes by the method of claim 9, wherein said cleavage results in a modification of said Rosa gene sequence by non-homologous end joining or homology directed repair.

15. The method of claim 14, wherein said modification comprises a deletion.

16. The method of claim 14, wherein said modification comprises insertion of an exogenous sequence.

17. A kit comprising the isolated fusion protein of claim 1.

18. A kit comprising the polynucleotide of claim 3.

19. The kit of claim 17, further comprising additional components selected from the group consisting of one or more exogenous sequences, instructions for use, and combinations thereof.

20. The kit of claim 18, further comprising additional components selected from the group consisting of one or more exogenous sequences, instructions for use, and combinations thereof.