Genetically-modified T cells comprising a modified intron in the T cell receptor alpha gene
The present invention provides a genetically-modified T cell comprising in its genome a modified human T cell receptor alpha gene. The modified T cell receptor alpha gene comprises an exogenous sequence of interest inserted into an intron within the T cell receptor alpha gene that is positioned 5′ upstream of TRAC exon 1. The exogenous sequence of interest can comprise an exogenous splice acceptor site and/or a poly A signal, which disrupts expression of the T cell receptor alpha subunit. The sequence of interest can also include a coding sequence for a polypeptide, such as a chimeric antigen receptor. Additionally, the endogenous splice donor site and the endogenous splice acceptor site flanking the intron are unmodified and/or remain functional. The invention further provides compositions and methods for producing the genetically-modified cell, and populations of the cell, and methods for the treatment of a disease, such as cancer, using such cells.
1. An isolated genetically-modified human T cell comprising in its genome a modified human T cell receptor alpha gene, wherein said modified human T cell receptor alpha gene comprises an exogenous sequence of interest inserted into an intron within the human T cell receptor alpha gene that is positioned 5′ upstream of the T cell receptor alpha constant region (TRAC) exon 1, and wherein said exogenous sequence of interest comprises an exogenous splice acceptor site or an exogenous splice acceptor site and a poly A signal, and wherein an endogenous splice donor site and an endogenous splice acceptor site flanking said intron are unmodified, and wherein cell surface expression of an endogenous human T cell receptor is reduced when compared to an unmodified control cell.
2. The isolated genetically-modified human T cell of claim 1 , wherein said intron comprises SEQ ID NO: 3.
3. The isolated genetically-modified human T cell of claim 1 , wherein said exogenous sequence of interest comprises, from 5′ to 3′, an exogenous splice acceptor site, a 2A element or IRES element, a coding sequence for a protein of interest, and a polyA signal.
4. The isolated genetically-modified human T cell of claim 3 , wherein said 2A element is a T2A, a P2A, an E2A, or an F2A element.
5. The isolated genetically-modified human T cell of claim 3 , wherein said 2A element is a T2A element.
6. The isolated genetically-modified human T cell of claim 1 , wherein said sequence of interest comprises a coding sequence for a chimeric antigen receptor or an exogenous T cell receptor.
7. The isolated genetically-modified human T cell of claim 1 , wherein said exogenous sequence of interest is inserted into said intron at an engineered meganuclease recognition site.
8. An isolated population of genetically-modified human T cells comprising a plurality of said isolated genetically-modified human T cell of claim 1 .
9. A pharmaceutical composition useful for treatment of a disease in a subject in need thereof, wherein said pharmaceutical composition comprises a pharmaceutically-acceptable carrier and said isolated genetically-modified human T cell of claim 1 .
10. A method of treating a disease in a subject in need thereof, said method comprising administering to said subject said isolated genetically-modified human T cell of claim 1 .