DNA-binding proteins and uses thereof

• US 9,322,005 B2
• Filed: 10/28/2013
• Issued: 04/26/2016
• Est. Priority Date: 05/17/2010
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Gregory, Philip D., Miller, Jeffrey C., Paschon, David, Rebar, Edward J., Tan, Siyuan, Urnov, Fyodor, Zhang, Lei
• Primary Examiner(s): Acevedo, James H Alstrum
• Assistant Examiner(s): YANG, KAIPEEN E
• Application Number:
  US14/064,991
• Publication Number:
  US 20140134740A1
• Time in Patent Office: 911 Days
• US Class Current: 1/1
• 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 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12Y 301/21004 Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

Disclosed herein are polypeptides, polynucleotides encoding, cells and organisms comprising novel DNA-binding domains, including TALE DNA-binding domains. Also disclosed are methods of using these novel DNA-binding domains for modulation of gene expression and/or genomic editing of endogenous cellular sequences.

Claims

1. An isolated, non-naturally occurring DNA-binding polypeptide comprising:

two or more TALE-repeat units, the TALE repeat units comprising a repeat variable di-residue (RVD) wherein the RVD binds to a nucleotide in a target DNA sequence;

an N-cap polypeptide flanking the N-terminal portion of the TALE-repeat units, wherein the N-cap polypeptide comprises a fragment of a full length N-terminus region of a TALE protein, the fragment comprising residues between N+122 and N+137; and

a C-cap polypeptide flanking the C-terminal portion of the TALE-repeat units, wherein the C-cap polypeptide comprises a fragment of a full length C-terminus region of a TALE protein,

wherein the polypeptide binds to DNA.

2. The isolated polypeptide of claim 1, wherein at least one TALE-repeat unit comprises an atypical repeat variable di-residue (RVD).

3. The polypeptide of claim 2, wherein the polypeptide comprises an atypical RVD selected from the group consisting of IG, HG, NK, DI, EI, AI, CI, HI, KI, RI, YD, ED, RD, AD, KD, ND, HN, DK, AN, DH, AK, SN, IP, LA, YG, SG, VG and IA.

4. The polypeptide of claim 1, wherein the C-cap polypeptide is less than approximately 230 amino acids in length.

5. The polypeptide of claim 1, wherein the C-cap comprises a TALE repeat domain.

6. A fusion protein comprising the polypeptide of claim 1 and at least one functional domain.

7. The fusion protein of claim 6, wherein the functional domain is a transcriptional activator or a transcriptional repressor.

8. The fusion protein of claim 6, wherein the functional domain comprises a nuclease.

9. The fusion protein of claim 8, wherein the nuclease comprises at least one cleavage domain or cleavage half-domain from a TypeIIS endonuclease.

10. A host cell comprising a polypeptide according to claim 1.

11. A pharmaceutical composition comprising a polypeptide according to claim 1.

12. A method of modulating expression of an endogenous gene in a cell, the method comprising:

introducing into the cell a fusion protein according to claim 6, wherein the fusion protein comprises a TALE-repeat domain that binds to a target site in the endogenous gene and further wherein expression of the endogenous gene is modulated.

13. The method of claim 12, wherein the modulation comprises gene activation.

14. The method of claim 12, wherein the modulation comprises gene repression or inactivation.

15. The method of claim 12, wherein the fusion protein comprises a cleavage domain or cleavage half-domain and the endogenous gene is inactivated by cleavage.

16. The method of claim 15, wherein inactivation occurs via non-homologous end joining (NHEJ).

17. The method of claim 12, wherein the fusion protein is introduced as a polynucleotide encoding the fusion protein.

18. A method of modifying a region of interest in the genome of a cell, the method comprising:

introducing into the cell at least one fusion protein according to claim 8, wherein the fusion protein comprises a TALE-repeat domain that binds to a target site in the genome of the cell and the fusion protein cleaves the genome in the region of interest.

19. The method of claim 18, wherein the modifying comprises introducing a deletion in the region of interest.

20. The method of claim 14, wherein the modifying comprises introducing an exogenous nucleic acid into the region of interest, the method further comprising introducing the exogenous nucleic acid into the cell, wherein the exogenous nucleic acid is integrated into the region of interest by homologous recombination.

21. The method of claim 18, wherein the cell is a eukaryotic cell selected from the selected from the group consisting of a plant cell, an animal cell, a fish cell and a yeast cell.

22. The method of claim 18, wherein the fusion protein is introduced as a polynucleotide encoding the fusion protein.