Targeted disruption of T cell receptor genes using engineered zinc finger protein nucleases

• US 10,155,011 B2
• Filed: 01/07/2015
• Issued: 12/18/2018
• Est. Priority Date: 11/10/2009
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Therapeutics, Inc.
• Inventors: Bonini, Maria Chiara, Genovese, Pietro, Gregory, Philip D., Holmes, Michael C., Naldini, Luigi, Paschon, David, Provasi, Elena, Zhang, Lei
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US14/591,625
• Publication Number:
  US 20150164954A1
• Time in Patent Office: 1,441 Days
• CPC Class Codes: A61K 35/00 Medicinal preparations containing materials or reaction products thereof with undetermined constitution
• 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 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C07K 2319/95 containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
• CPC Class Codes: C12N 15/01 Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for inactivating TCR genes, using zinc finger nucleases (ZFNs) comprising a zinc finger protein and a cleavage domain or cleavage half-domain in conditions able to preserve cell viability. Polynucleotides encoding ZFNs, vectors comprising polynucleotides encoding ZFNs and cells comprising polynucleotides encoding ZFNs and/or cells comprising ZFNs are also provided. Disclosed herein are also methods and compositions for expressing a functional exogenous TCR in the absence of endogenous TCR expression in T lymphocytes, including lymphocytes with a central memory phenotype. Polynucleotides encoding exogenous TCR, vectors comprising polynucleotides encoding exogenous TCR and cells comprising polynucleotides encoding exogenous TCR and/or cells comprising exogenous TCR are also provided.

Claims

1. An isolated genetically modified cell comprising a genomic modification that modifies the endogenous T-cell receptor (TCR) gene, wherein the genomic modification is selected from the group consisting of insertions, deletions and combinations thereof, wherein the genomic modification is produced by a zinc finger nuclease comprising a zinc finger protein that binds to a target site in the TCR gene, wherein said target site is any of SEQ ID NO:

1, 7, 13, 18, 23, 29, 34, 37, 41, 45, 49, 54, 58 or 63, wherein the binding of the zinc finger protein at the target site in the TCR gene results in cleavage of the TCR gene such that the TCR gene is modified within any of SEQ ID NO;

1, 7, 13, 18, 23, 29, 34, 37, 41, 45, 49, 54, 58 and 63, thereby resulting in a genetically modified cell comprising the genomic modification at the endogenous TCR gene.

2. The genetically modified cell of claim 1, wherein the genomic modification is within an endogenous TCRα

or TCRβ

gene.

3. The genetically modified cell of claim 2, wherein the genomic modification is within exon 1 or exon 3 of an endogenous TCRα

gene.

4. The genetically modified cell of claim 1, wherein the endogenous TCR gene is inactivated.

5. The genetically modified cell of claim 1, wherein the cell is a T-cell.

6. The genetically modified cell of claim 1, wherein the cell is a stem cell.

7. The genetically modified cell of claim 6, wherein the stem cell is a hematopoietic stem cell.

8. A genetically modified differentiated cell descended from the stem cell of claim 6.

9. The isolated genetically modified cell of claim 1, wherein the zinc finger nuclease is introduced into the cell as a polynucleotide.

10. The genetically modified cell of claim 1, further comprising a donor polynucleotide encoding a transgene.

11. The genetically modified cell of claim 10, wherein the transgene encodes an engineered T cell receptor subunit.

12. The genetically modified cell of claim 10, wherein the transgene encodes a chimeric antigen receptor (CAR).

13. A pharmaceutical composition comprising the genetically modified cell of claim 1.