Materials and methods for engineering cells and uses thereof in immuno-oncology
AssigneeCrispr Therapeutics AG
InventorsJonathan Alexander TERRETT, Demetrios KALAITZIDIS, Lawrence KLEIN
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Abstract
Materials and methods for producing genome-edited cells engineered to express a chimeric antigen receptor (CAR) construct on the cell surface, and materials and methods for genome editing to modulate the expression, function, or activity of one or more immuno-oncology related genes in a cell, and materials and methods for treating a patient using the genome-edited engineered cells.
Claims31
-186. (canceled)
A population of cells comprising engineered human T cells, wherein the engineered human T cells comprise:
- (a) a disrupted T cell receptor alpha chain constant region (TRAC) gene comprising a deletion of the nucleotide sequence of SEQ ID NO:
- 76, and comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising (i) an ectodomain that comprises an anti-CD70 single-chain variable fragment (scFv), wherein the anti-CD70 scFv comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:
- 1592, and a variable light chain comprising the amino acid sequence of SEQ ID NO:
- 1593, (ii) a CD8 transmembrane domain, and (iii) an endodomain that comprises a CD28 or 41BB co-stimulatory domain and a CD3z co-stimulatory domain;
- and
- (b) a disrupted beta-2-microglobulin (B2M) gene.
A population of cells comprising engineered human T cells, wherein the engineered human T cells comprise:
- (a) a disrupted TRAC gene comprising a deletion of the nucleotide sequence of SEQ ID NO:
- 76, and comprising a nucleic acid encoding a CAR comprising (i) an ectodomain that comprises an anti-CD70 scFv, wherein the anti-CD70 scFv comprises the amino acid sequence of SEQ ID NO:
- 1499 or SEQ ID NO:
- 1500, (ii) a CD8 transmembrane domain, and (iii) an endodomain that comprises a CD28 or 41BB co-stimulatory domain and a CD3z co-stimulatory domain;
- and
- (b) a disrupted B2M gene.
A population of cells comprising engineered human T cells, wherein the engineered human T cells comprise:
- (a) a disrupted TRAC gene comprising a deletion of the nucleotide sequence of SEQ ID NO:
- 76, and comprising a nucleic acid encoding a CAR, wherein the CAR comprises the amino acid sequence of SEQ ID NO:
- 1449, 1450, or 1276;
- and
- (b) a disrupted B2M gene.
A method for producing an engineered human T cell, the method comprising delivering to a human T cell:
- (a) a RNA-guided nuclease;
- (b) a gRNA targeting a TRAC gene;
- (c) a gRNA targeting a B2M gene;
- and
- (d) a vector comprising a nucleic acid comprising (i) a nucleotide sequence that comprises a left region of homology to the TRAC locus, (ii) a nucleotide sequence encoding an anti-CD70 CAR, and (iii) a nucleotide sequence that comprises a right region of homology to the TRAC gene locus,
- thereby producing an engineered human T cell.
Dependencies are inferred from the claim text.