Methods and compositions for gene inactivation

• US 20080159996A1
• Filed: 05/23/2007
• Published: 07/03/2008
• Est. Priority Date: 05/25/2006
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Holmes, Michael C., Ando, Dale, Lee, Gary Ka Leong
• Granted Patent: US 7,951,925 B2
• Time in Patent Office: Days
• US Class Current: 424/93.21
• 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 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 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, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F3, and F4 comprise the following amino acid sequences:

2. The protein according to claim 1, wherein F2 comprises the amino acid sequence ISSNLNS (SEQ ID NO:

5).

3. The protein according to claim 1, wherein F2 comprises the amino acid sequence VSSNLTS (SEQ ID NO:

6).

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

5. The protein of claim 4, wherein the cleavage domain is a cleavage half-domain.

6. The protein of claim 5, wherein the cleavage half-domain is a wild-type FokI cleavage half-domain.

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

8. A polynucleotide encoding the protein of claim 1.

9. A gene delivery vector comprising a polynucleotide according to claim 8.

10. The gene delivery vector of claim 9, wherein the vector is an adenovirus vector.

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

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

13. An isolated cell comprising the polynucleotide of claim 8.

14. 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.

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

16. The cell of claim 13, 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.

17. The cell of claim 16, wherein the T-cell is a CD4⁺ cell.

18. A method for inactivating the CCR-5 gene in a human cell, the method comprising administering to the cell a polynucleotide according to claim 8.

19. The method of claim 18, 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.

20. The method of claim 19, wherein the T-cell is a CD4⁺ cell.

21. A method for treating or preventing HIV infection in a subject, the method comprising:

(a) introducing, into a cell, a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises;

(i) a zinc finger DNA-binding domain that is engineered to bind to a first target site in the CCR5 gene; and

(ii) a cleavage domain;

such that the polypeptide is expressed in the cell, whereby the polypeptide binds to the target site and cleaves the CCR5 gene; and

(b) introducing the cell into the subject,

and further 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 nucleic acid comprises 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 F1, F3, and F4 comprise the following amino acid sequences:

23. The method of claim 21, wherein the first nucleic acid further encodes a second polypeptide, wherein the second polypeptide comprises:

(i) a zinc finger DNA-binding domain that is engineered to bind to a second target site in the CCR5 gene; and

(ii) a cleavage domain;

such that the second polypeptide is expressed in the cell, whereby the first and second polypeptides bind to their respective target sites and cleave the CCR5 gene.

24. The method of claim 21, further comprising the step of introducing into the cell a second nucleic acid, wherein the second nucleic acid contains two regions of homology to the CCR-5 gene, flanking a sequence that is non-homologous to the CCR-5 gene.