Altering gene expression in modified T cells and uses thereof
The present invention relates to compositions and methods for generating a modified T cell with a nucleic acid capable of downregulating endogenous gene expression selected from the group consisting of TCR α chain, TCR β chain, beta-2 microglobulin and FAS further comprising a nucleic acid encoding a modified T cell receptor (TCR) comprising affinity for a surface antigen on a target cell or an electroporated nucleic acid encoding a chimeric antigen receptor (CAR). Also included are methods and pharmaceutical compositions comprising the modified T cell for adoptive therapy and treating a condition, such as an autoimmune disease.
1. A method for generating a CRISPR-modified T cell, the method comprising:
introducing into a T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous TCR α chain in the T cell, wherein the nucleic acid that causes downregulation of gene expression of the endogenous TCR α chain comprises a nucleotide sequence encoded by SEQ ID NO: 1;
introducing into the T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous TCR β chain in the T cell; and
introducing into the T cell a nucleic acid encoding an 8F T cell receptor (TCR) comprising affinity for NY-ESO-1 on a target cell,
thereby generating the CRISPR-modified T cell.
2. The method of claim 1 , further comprising introducing into a T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous beta-2-microglobulin gene.
3. The method of claim 1 , wherein the CRISPR system comprises an pAd5/F35-CRISPR vector.
4. The method of claim 1 , wherein the T cell is obtained from the group consisting of peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line.
5. The method of claim 1 , wherein the method further comprises expanding the CRISPR-modified T cell.
6. The method of claim 5 , wherein the expanding comprises electroporating the T cell with RNA encoding a chimeric membrane protein, thereby generating an electroporated T cell and culturing the electroporated T cell.
7. The method of claim 6 , wherein the culturing is conducted at about 32° C.
8. The method of claim 6 , wherein the chimeric membrane protein comprises a single chain variable fragment (scFv) against CD3 and an intracellular domain comprising a portion of an intracellular domain of CD28 and a portion of an intracellular signaling domain of 4-IBB.
9. The method of claim 5 , wherein the expanding comprises culturing the CRISPR-modified T cell with a factor selected from the group consisting of flt3-L, IL-1, IL-3, and c-kit ligand.
10. The method of claim 1 , further comprising cryopreserving the T cell prior to introducing the nucleic acids into the T cell, thereby generating a cryopreserved T cell.
11. The method of claim 1 , further comprising thawing the cryopreserved T cell prior to introducing the nucleic acids into the T cell.
12. The method of claim 1 , wherein introducing the nucleic acids is conducted by a method selected from the group consisting of transducing the T cell, transfecting the T cell, and electroporating the T cell.
13. The method of claim 1 , further comprising electroporating a RNA encoding a co-stimulatory molecule into the T cell.
14. The method of claim 1 , further comprising expressing Klf4, Oct3/4, and Sox2 in the T cells to induce pluripotency of the T cell.
15. A method for generating a CRISPR-modified T cell, the method comprising:
introducing into a T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous TCR α chain in the T cell, wherein the nucleic acid that causes downregulation of gene expression of the endogenous TCR α chain comprises a nucleotide sequence encoded by SEQ ID NO: 1;
introducing into the T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous TCR β chain in the T cell;
introducing into the T cell a CRISPR system comprising a nucleic acid that causes downregulation of gene expression of an endogenous PD1 gene in the T cell; and
introducing into the T cell a nucleic acid encoding an 8F T cell receptor (TCR) comprising affinity for NY-ESO-1 on a target cell,
thereby generating the CRISPR-modified T cell.
16. A method for generating a CRISPR-modified T cell, the method comprising introducing into a T cell:
i) a first nucleic acid encoding Cas9;
ii) a second nucleic acid comprising a nucleotide sequence encoded by SEQ ID NO: 1;
iii) a third nucleic acid; and
iv) a fourth nucleic acid encoding an 8F T cell receptor (TCR), the TCR comprising affinity for NY-ESO-1;
wherein, in the introducing:
i) Cas9 is expressed;
ii) a nucleotide sequence of the second nucleic acid binds to the Cas9 and the nucleotide sequence of SEQ ID NO: 1 hybridizes with an endogenous TCR α chain gene,
thereby causing downregulation of the endogenous TCR α gene; and
iii) a nucleotide sequence of the third nucleic acid binds to the Cas9 and hybridizes with an endogenous TCR β chain gene, thereby causing downregulation of the endogenous TCR β gene;
thereby generating the CRISPR-modified T cell.