Methods and compositions for gene inactivation

• US 8,524,221 B2
• Filed: 05/05/2011
• Issued: 09/03/2013
• 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., Jouvenot, Yann, Lee, Gary Ka Leong
• Primary Examiner(s): LI, QIAN JANICE
• Application Number:
  US13/068,230
• Publication Number:
  US 20120308528A1
• Time in Patent Office: 852 Days
• Field of Search: None
• US Class Current: 424/93.2
• 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 method for inactivating a CCR-5 gene in an isolated CD34+ cell, the method comprising administering to the cell a polynucleotide encoding a first fusion protein comprising a first cleavage domain and a first 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 method of claim 1, wherein F2 comprises the amino acid sequence ISSNLNS (SEQ ID NO:

5).

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

6).

4. The method of claim 1, wherein the first cleavage domain comprises a FokI cleavage domain.

5. The method of claim 1, further comprising the step of administering a viral vector encoding a second fusion protein comprising a second cleavage domain and a second engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition helices as follows:

6. The method of claim 5, wherein the second cleavage domain comprises a FokI cleavage domain.

7. The method of claim 1, wherein the polynucleotide comprises a viral vector.

8. The method of claim 7, where the viral vector is a lentiviral vector, an adenoviral vector, or an adeno-associated vector.

9. The method of claim 7, wherein the viral vector encodes the first and second fusion proteins.

10. The method of claim 1 wherein the polynucleotide comprises a plasmid or RNA.

11. The method of claim 1, wherein the CD34+ cell is a human cell.

12. A method of treating or preventing HIV infection in a subject, the method comprising:

inactivating a CCR5 gene in an isolated CD34+ cell according to the method of claim 1;

introducing the isolated CD34+ cell into the subject.

13. The method of claim 12, wherein the subject is a human.