CPC A61K 35/17 (2013.01) [C07K 14/7051 (2013.01); C12N 5/0636 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/625 (2013.01); C12N 15/907 (2013.01); A61K 38/00 (2013.01); C07K 2319/00 (2013.01); C12N 2310/20 (2017.05); C12N 2501/2302 (2013.01); C12N 2501/2307 (2013.01); C12N 2501/2315 (2013.01); C12N 2800/80 (2013.01)] | 26 Claims |
1. A method for making a population of engineered T cells, the method comprising:
a) contacting a population of T cells with a first ribonucleoprotein particle (RNP) and a donor DNA, wherein the first RNP comprises a first guide RNA that targets an endogenous TCR locus, and wherein the donor DNA comprises a nucleic acid sequence comprising a gene encoding a polypeptide comprising an exogenous TCR-beta and an exogenous TCR-alpha or portion thereof, under conditions to allow the RNP and the donor DNA to enter the T cells;
b) incubating the population of T cells for a period of time; and
c) culturing the population of T cells in a medium for a period of time to allow the gene to be inserted into the endogenous TCR locus, thereby forming a population of engineered T cells
wherein the amount of donor DNA is about 0.0004 pmol/μL to about 0.4 pmol/μL,
wherein the donor DNA is on a nanoplasmid,
and wherein the efficiency of the gene insertion is at least 27%.
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