Genetically-modified cells comprising a modified human T cell receptor alpha constant region gene
Disclosed herein is a genetically-modified cell comprising in its genome a modified human T cell receptor alpha constant region gene, wherein the cell has reduced cell-surface expression of the endogenous T cell receptor. The present disclosure further relates to methods for producing such a genetically-modified cell, and to methods of using such a cell for treating a disease in a subject.
1. A method for producing a genetically-modified human T cell comprising a modified human TCR alpha constant region gene, said method comprising:
(a) introducing into a human T cell an mRNA comprising a first nucleic acid sequence encoding an engineered nuclease,
wherein said engineered nuclease produces a cleavage site at a recognition sequence within said human TCR alpha constant region gene; and
(b) introducing into said human T cell a second nucleic acid sequence comprising an exogenous polynucleotide encoding a chimeric antigen receptor comprising an extracellular ligand-binding domain, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain, or encoding an exogenous T cell receptor,
wherein said second nucleic acid sequence is introduced by contacting said human T cell with a recombinant adeno-associated virus (AAV) vector comprising said second nucleic acid sequence;
wherein said second nucleic acid sequence comprises, from 5′ to 3′:
(i) a 5′ homology arm that is homologous to the 5′ upstream sequence flanking said cleavage site;
(ii) said exogenous polynucleotide; and
(iii) a 3′ homology arm that is homologous to the 3′ downstream sequence flanking said cleavage site;
wherein the sequence of said exogenous polynucleotide is inserted into said human TCR alpha constant region gene at said cleavage site by homologous recombination,
and further wherein said genetically-modified human T cell does not express an endogenous TCR on the cell surface.
2. The method of claim 1 , wherein said recombinant AAV vector is a single-strand AAV vector.
3. The method of claim 1 , wherein said recombinant AAV vector has a serotype of AAV6.
4. The method of claim 1 , wherein said recombinant AAV vector has a serotype of AAV2.
5. The method of claim 1 , wherein said engineered nuclease is an engineered meganuclease, a recombinant zinc-finger nuclease (ZFN), a recombinant transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease.
6. The method of claim 5 , wherein said engineered nuclease is an engineered meganuclease.
7. The method of claim 6 , wherein said recognition sequence within said human TCR alpha constant region gene consists of SEQ ID NO: 3.
8. The method of claim 1 , wherein said exogenous polynucleotide comprises a promoter that drives expression of said chimeric antigen receptor or said exogenous T cell receptor.
9. The method of claim 1 , wherein said recombinant AAV vector is a single-strand AAV vector, and wherein said recombinant AAV vector has a serotype of AAV6 or AAV2.
10. The method of claim 9 , wherein said engineered nuclease is an engineered meganuclease, a recombinant zinc-finger nuclease (ZFN), a recombinant transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease.
11. The method of claim 10 , wherein said engineered nuclease is an engineered meganuclease.
12. The method of claim 11 , wherein said recognition sequence within said human TCR alpha constant region gene consists of SEQ ID NO: 3.
13. The method of claim 1 , wherein said recombinant AAV vector is a single-strand AAV vector, and wherein said recombinant AAV vector has a serotype of AAV6 or AAV2, and wherein said exogenous polynucleotide comprises a promoter that drives expression of said chimeric antigen receptor or said exogenous T cell receptor.
14. The method of claim 13 , wherein said engineered nuclease is an engineered meganuclease, a recombinant zinc-finger nuclease (ZFN), a recombinant transcription activator-like effector nuclease (TALEN), a CRISPR/Cas nuclease, or a megaTAL nuclease.
15. The method of claim 14 , wherein said engineered nuclease is an engineered meganuclease.
16. The method of claim 15 , wherein said recognition sequence within said human TCR alpha constant region gene consists of SEQ ID NO: 3.
17. The method of claim 1 , wherein said intracellular co-stimulatory domain is a 4-1BB co-stimulatory domain.