US 12,344,843 B2
Altering gene expression in cart cells and uses thereof
Yangbing Zhao, Lumberton, NJ (US); Jiangtao Ren, Philadelphia, PA (US); Xiaojun Liu, Wallingford, PA (US); and Carl H. June, Merion Station, PA (US)
Assigned to The Trustees of the University of Pennsylvania, Philadelphia, PA (US)
Filed by THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA, Philadelphia, PA (US)
Filed on Jul. 30, 2020, as Appl. No. 16/943,335.
Application 16/943,335 is a continuation of application No. 15/516,240, abandoned, previously published as PCT/US2015/055799, filed on Oct. 15, 2015.
Claims priority of provisional application 62/073,651, filed on Oct. 31, 2014.
Prior Publication US 2020/0407728 A1, Dec. 31, 2020
Int. Cl. C12N 5/0783 (2010.01); A61K 35/17 (2015.01); A61K 35/26 (2015.01); A61K 39/00 (2006.01); A61K 40/11 (2025.01); A61K 40/22 (2025.01); A61K 40/31 (2025.01); A61K 40/32 (2025.01); A61K 40/36 (2025.01); A61K 40/41 (2025.01); A61K 40/42 (2025.01); A61P 35/00 (2006.01); A61P 37/06 (2006.01); C12N 5/10 (2006.01); C12N 15/113 (2010.01); C12N 15/12 (2006.01); C12N 15/62 (2006.01); C12N 15/85 (2006.01); A61K 40/50 (2025.01)
CPC C12N 15/1138 (2013.01) [A61K 35/17 (2013.01); A61K 35/26 (2013.01); A61K 39/001102 (2018.08); A61K 40/11 (2025.01); A61K 40/22 (2025.01); A61K 40/31 (2025.01); A61K 40/32 (2025.01); A61K 40/36 (2025.01); A61K 40/416 (2025.01); A61K 40/418 (2025.01); A61K 40/4211 (2025.01); A61K 40/4269 (2025.01); A61K 40/4274 (2025.01); A61P 35/00 (2018.01); A61P 37/06 (2018.01); C12N 5/0636 (2013.01); C12N 15/85 (2013.01); A61K 2039/5156 (2013.01); A61K 2039/5158 (2013.01); A61K 40/50 (2025.01); C12N 2310/10 (2013.01); C12N 2310/20 (2017.05); C12N 2501/48 (2013.01); C12N 2501/515 (2013.01); C12N 2501/599 (2013.01); C12N 2501/998 (2013.01); C12N 2510/00 (2013.01)] 8 Claims
 
1. A CRISPR-modified T cell comprising:
(i) a CRISPR-mediated insertion or deletion in an endogenous TCR a chain (TRAC) and an endogenous TCR β chain (TRBC) gene locus causing downregulated gene expression of the endogenous TRAC gene and the endogenous TRBC gene;
(ii) a CRISPR-mediated insertion or deletion in an endogenous beta 2-microglobulin (B2M) gene locus causing downregulated gene expression of the endogenous B2M gene; and
(iii) a nucleic acid encoding a chimeric antigen receptor (CAR) having an affinity for a tumor associated-antigen (TAA) on a target cell, wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain;
wherein the CRISPR system that mediates the insertion or deletion in the endogenous TRAC gene, the endogenous TRBC gene, or the endogenous B2M gene comprises a CRISPR-associated (Cas) nuclease and:
(a) a first guide RNA comprising a nucleic acid sequence capable of targeting the sequence of SEQ ID NO: 15 in the coding sequence of the TRAC gene;
(b) a second guide RNA comprising a nucleic acid sequence capable of targeting the sequence of SEQ ID NO: 16 in the coding sequence of the TRBC gene; and
(c) a third guide RNA comprising a nucleic acid sequence capable of targeting the sequence of SEQ ID NO: 42 in the coding sequence of the B2M gene.