US 12,239,664 B2
Efficient TCR gene editing in T lymphocytes
Sascha Rutz, San Francisco, CA (US); Benjamin Joseph Haley, San Francisco, CA (US); Shravan Madireddi, Fremont, CA (US); Soyoung Oh, San Bruno, CA (US); David Shaw, Burlingame, CA (US); and Kate Halliwell Senger, Oakland, CA (US)
Assigned to GENENTECH, INC., South San Francisco, CA (US)
Filed by Genentech, Inc., South San Francisco, CA (US)
Filed on Mar. 24, 2022, as Appl. No. 17/703,927.
Claims priority of provisional application 63/323,065, filed on Mar. 24, 2022.
Claims priority of provisional application 63/165,509, filed on Mar. 24, 2021.
Prior Publication US 2023/0041268 A1, Feb. 9, 2023
Int. Cl. A61K 35/17 (2015.01); A61K 38/00 (2006.01); C07K 14/725 (2006.01); C12N 5/0783 (2010.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/62 (2006.01); C12N 15/90 (2006.01)
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%.