Methods and compositions for gene inactivation

• US 9,434,776 B2
• Filed: 10/15/2013
• Issued: 09/06/2016
• Est. Priority Date: 05/25/2006
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Ando, Dale, Holmes, Michael C., Lee, Gary Ka Leong
• Primary Examiner(s): LI, QIAN JANICE
• Application Number:
  US14/054,042
• Publication Number:
  US 20140127811A1
• Time in Patent Office: 1,057 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 2239/31 characterized by the route of administration
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• 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: A61K 39/4611 T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
• CPC Class Codes: A61K 39/464838 Viral antigens
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: C07K 14/47 from mammals
• CPC Class Codes: C07K 14/4703 Inhibitors; Suppressors
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2710/10345 Special targeting system for viral vectors
• CPC Class Codes: C12N 5/10 Cells modified by introduction of foreign genetic material
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for inactivating CCR-5 genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs, such as adenovirus (Ad) vectors, and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided.

Claims

1. A protein comprising an engineered zinc finger protein DNA-binding domain that binds to an endogenous CCR5 gene, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and further wherein the zinc finger DNA-binding domains comprise recognition regions selected from the group consisting of:

2. A protein according to claim 1, further comprising a cleavage domain.

3. The protein of claim 2, wherein the cleavage domain is a cleavage half-domain.

4. The protein of claim 3, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

5. The protein of claim 3, wherein the cleavage half-domain is an engineered FokI cleavage half-domain.

6. A polynucleotide encoding the protein of claim 1.

7. A gene delivery vector comprising a polynucleotide according to claim 6.

8. The gene delivery vector of claim 7, wherein the vector is an adenovirus vector.

9. The gene delivery vector of claim 8, wherein the adenovirus vector is an Ad5/35 vector.

10. An isolated cell comprising the protein of claim 1.

11. The isolated cell of claim 10, further comprising a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein zinc finger DNA-binding domains comprise recognition regions selected from the group consisting of:

12. An isolated cell comprising the polynucleotide of claim 6.

13. The isolated cell of claim 12, further comprising a polynucleotide encoding a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein zinc finger DNA-binding domains comprise recognition regions selected from the group consisting of:

14. The cell of claim 10, wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.

15. The cell of claim 14, wherein the T-cell is a CD4⁺ cell.

16. The cell of claim 14, wherein the hematopoietic stem cell is a CD34+ cell.

17. The cell of claim 12, wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.

18. The cell of claim 17, wherein the T-cell is a CD4⁺ cell.

19. A method for inactivating the CCR-5 gene in a human cell, the method comprising administering to the cell a polynucleotide according to claim 6.

20. The method of claim 19, further comprising administering a polynucleotide encoding a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein zinc finger DNA-binding domains comprise recognition regions selected from the group consisting of:

21. The method of claim 19, wherein the cell is selected from the group consisting of a hematopoietic stem cell, a T-cell, a macrophage, a dendritic cell and an antigen-presenting cell.

22. The method of claim 21, wherein the T-cell is a CD4⁺ cell.

23. The cell of claim 21, wherein the hematopoietic stem cell is a CD34+ cell.