DNA-binding proteins and uses thereof

• US 9,493,750 B2
• Filed: 10/28/2013
• Issued: 11/15/2016
• 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): Leith, Nancy J
• Application Number:
  US14/065,028
• Publication Number:
  US 20140134723A1
• Time in Patent Office: 1,114 Days
• Field of Search: None
• 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 polynucleotide encoding a non-naturally occurring fusion protein comprising:

(i) at least one functional domain, wherein the functional domain comprises a transcriptional activator, a transcriptional repressor or a nuclease; and

(ii) a 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 at least one TALE-repeat unit is non-naturally occurring;

an N-cap polypeptide, wherein the N-cap comprises a fragment of a full length N-terminus region of a TALE protein and does not comprise a full length N-terminal region of a TALE protein, the N-cap comprising residues N+1 to N+121 of a TALE protein; and

a C-cap polypeptide, wherein the C-cap polypeptide comprises a fragment of a full length C-terminus region of a TA LE protein and does not comprise a full length C-terminal region of a TALE protein, the C-cap comprising residues C-1 to C-20 of the TALE protein, wherein the polypeptide binds to DNA.

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

3. The polynucleotide of claim 2, wherein the protein comprises an atypical RVD as shown in Tables 27A or 27B.

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

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

6. The polynucleotide of claim 1, wherein the functional domain is a transcriptional activator or a transcriptional repressor.

7. The polynucleotide of claim 1, wherein the functional domain comprises a nuclease.

8. The polynucleotide of claim 7, wherein the nuclease comprises at least one cleavage domain or cleavage half-domain from a TypeIIS endonuclease.

9. An isolated host cell comprising a polynucleotide according to claim 1.

10. A pharmaceutical composition comprising a polynucleotide according to claim 1.