Methods and systems for engineering cells and for target validation
AssigneeBristol-Myers Squibb Company
InventorsEugene DROKHLYANSKY, Chao DAI, Brian Anthony RABE, Anna LYUBETSKAYA
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Abstract
Provided are vectors, systems, and methods for gene editing that result in reduced immunogenicity in a host.
Claims88
A vector comprising i) a nucleic acid encoding a Cas enzyme and/or or a nucleic acid encoding a guide RNA and ii) a nucleic acid encoding a selectable marker, wherein the nucleic acid encoding the selectable marker is flanked by recognition sites for a site-specific recombinase and wherein the nucleic acid encoding the Cas enzyme or the nucleic acid encoding the guide RNA is located outside of the recognition sites for the site-specific recombinase.
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A method of generating cells comprising a nucleic acid encoding a Cas enzyme comprising
- removing nucleic acid encoding a selectable marker from a vector within a plurality of cells, wherein the vector comprises a nucleic acid encoding a Cas enzyme, a nucleic acid encoding a guide RNA, and the nucleic acid encoding the selectable marker, wherein the resulting cells comprise engineered cells comprising the nucleic acid encoding the Cas enzyme.
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A method of introducing engineered cells comprising a Cas enzyme and a guide RNA into an organism that has an immune response, comprising
- generating the engineered cells by
- providing one or more vectors comprising the Cas enzyme, the guide RNA, and a selectable marker to a plurality of cells,
- culturing the cells such that the plurality of cells express the selectable marker,
- isolating from the cultured cells, selected cells the express the selectable marker,
- removing the selectable marker from the isolated, selected cells to produce engineered cells,
- providing the engineered cells to the organism, wherein the organism is tolerized to the Cas enzyme.
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Dependencies are inferred from the claim text.