Targeted disruption of T cell receptor genes using talens

• US 9,937,207 B2
• Filed: 03/20/2014
• Issued: 04/10/2018
• Est. Priority Date: 03/21/2013
• Status: Active Grant

• Current Assignee: Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Therapeutics, Inc.
• Original Assignee: Ospedale San Raffaele Srl (Policlinico San Donato S.p.A.), Sangamo Therapeutics, Inc.
• Inventors: Gregory, Philip D., Holmes, Michael C., Paschon, David, Zhang, Lei, Bonini, Maria Chiara, Genovese, Pietro, Magnani, Zulma, Mastaglio, Sara, Naldini, Luigi
• Primary Examiner(s): Moloye, Titilayo
• Application Number:
  US14/221,074
• Publication Number:
  US 20140301990A1
• Time in Patent Office: 1,482 Days
• CPC Class Codes: A61K 2239/38 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 2239/48 Blood cells, e.g. leukemia or lymphoma
• CPC Class Codes: A61K 35/26 Lymph; Lymph nodes; Thymus; Spleen; Splenocytes; Thymocytes
• 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 39/4632 T-cell receptors [TCR]; antibody T-cell receptor constructs
• CPC Class Codes: A61K 39/464453 Wilms tumor 1 [WT1]
• CPC Class Codes: A61K 39/464488 NY-ESO
• 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: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2501/515 CD3, T-cell receptor complex
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2740/16043 viral genome or elements thereof as genetic vector

Abstract

Disclosed herein are methods and compositions for modifying TCR genes, using nucleases (zinc finger nucleases or TAL nucleases) to modify TCR genes.

Claims

1. An isolated T-lymphocyte comprising a stably integrated exogenous sequence encoding a T-cell receptor (TCR), wherein at least one endogenous TCR gene within the cell is partially or completely inactivated by a nuclease, wherein the nuclease is a TALEN that binds to a target sequence site in the endogenous TCR gene, wherein said target sequence site is selected from SEQ ID NO:

147-153, the TALEN comprising a TAL-effector DNA-binding domain and a cleavage domain, and further wherein the TAL-effector DNA-binding domain comprises a C-terminal truncation as compared to a wild-type TAL-effector DNA-domain.

2. The isolated T-lymphocyte of claim 1, wherein the endogenous TCR gene is a TCR α

or TCR β

gene.

3. The isolated T-lymphocyte of claim 1, wherein a polynucleotide encoding the TALEN that binds to a target sequence site selected from SEQ ID NO:

147-153 is introduced into the cell using an integrase-defective lentiviral vector (IDLV), AAV, a plasmid or mRNA, the TALEN comprising a TAL-effector DNA-binding domain and a cleavage domain, and further wherein the TAL-effector DNA-binding domain comprises a C-terminal truncation as compared to a wild-type TAL-effector DNA-domain.

4. The isolated T-lymphocyte of claim 1, wherein the exogenous sequence is introduced into an endogenous TCR gene, a CCR5 gene or an AAVS1 gene.

5. The isolated T-lymphocyte of claim 1, wherein the exogenous sequence is selected from the group consisting of a tumor antigen specific TCR transgene wherein the TCR transgene is a TCR α

transgene, a TCR β

transgene and combinations thereof.

6. The isolated T-lymphocyte of claim 5, wherein the tumor antigen comprises NY-ESO1.

7. A pharmaceutical composition comprising the isolated T-lymphocyte of claim 1.

8. A method of generating a T-lymphocyte according to claim 1, the method comprising:

inactivating an endogenous TCR gene in the T-lymphocyte using one or more polynucleotides encoding the one or more TALENs, each TALEN comprising a TAL-effector DNA-binding domain and a cleavage domain, and further wherein the TAL-effector DNA-binding domain comprises a C-terminal truncation as compared to a wild-type TAL-effector DNA-domain, wherein the TALEN cleave the endogenous TCR gene; and

stably integrating the exogenous sequence into the genome of the T-lymphocyte.

9. The method of claim 8, wherein the endogenous TCR gene is a TCR α

and/or TCR β

gene.

10. The method of claim 8, wherein the exogenous sequence is introduced into the cell using an integrase-defective lentiviral vector (IDLV), retroviral vector (RV) or lentiviral Vector (LV).

11. The method of claim 10, wherein the exogenous sequence is introduced by IDLV into an endogenous TCR gene, a CCR5 gene or an AAVS1 gene.

12. The method of claim 8, wherein the exogenous sequence is selected from the group consisting of a tumor antigen specific TCR transgene wherein the TCR transgene is a TCR α

transgene, a TCR β

transgene and combinations thereof.

13. The method of claim 12, wherein the tumor antigen comprises NY-ESO1.