Modification of CXCR4 using engineered zinc finger proteins

• US 9,717,759 B2
• Filed: 09/24/2014
• Issued: 08/01/2017
• Est. Priority Date: 03/20/2009
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Holmes, Michael C., Miller, Jeffrey C., Wang, Jianbin
• Primary Examiner(s): HILL, KEVIN KAI
• Application Number:
  US14/495,529
• Publication Number:
  US 20150110757A1
• Time in Patent Office: 1,042 Days
• Field of Search: 424 932, 424 941, 435455, 435462, 514 44, 530350, 536 234
• 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 35/17 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 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: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 14/7158 for chemokines
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding

Abstract

Disclosed herein are methods and compositions for modulating activity of CXCR4 genes, for example using zinc finger transcription factors (ZF-TFs) or zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding ZF-TFs or ZFNs, vectors comprising polynucleotides encoding ZF-TFs or ZFNs and cells comprising polynucleotides encoding ZF-TFs or ZFNs and/or cells comprising ZF-TF or ZFNs are also provided.

Claims

1. An isolated genetically modified cell comprising a genomic modification within exon 2 of a CXCR4 gene, wherein the genomic modification is introduced using a nuclease that binds to a target site as shown in any of SEQ ID NO:

1, SEQ ID NO;

6, SEQ ID NO;

11, SEQ ID NO;

16, SEQ ID NO;

21 or SEQ ID NO;

25, the genomic modification comprising a deletion, wherein the CXCR4 gene is inactivated and further wherein if the genomic modification further comprises an insertion, the insertion is a non-coding sequence of less than 3 base pairs.

2. The genetically modified cell of claim 1, wherein the genetic modification is within an endogenous sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

6, SEQ ID NO;

11, SEQ ID NO;

16, SEQ ID NO;

21 and SEQ ID NO;

25.

3. The genetically modified cell of claim 1, 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.

4. The genetically modified cell of claim 1, wherein the modification is introduced using at least one zinc finger nuclease.

5. The genetically modified cell of claim 4, wherein the nuclease is introduced into the cell as a polynucleotide.

6. The genetically modified cell of claim 4, wherein the zinc finger nuclease comprises 4 or 5 zinc finger domains comprising a recognition helix and further wherein the zinc finger proteins comprise the recognition helix regions in the order shown in a single row of Table 1.

7. The genetically modified cell of claim 1, wherein an endogenous CCR5 gene is inactivated in the cell.

8. The genetically modified cell of claim 1, wherein the cell is resistant to HIV infection.

9. The genetically modified cell of claim 7, wherein the cell is resistant to HIV infection.

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

introducing a cell according to claim 1 into the subject.

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

introducing a cell according to claim 7 into the subject.