Non-viral intron knock-ins for targeted gene integration into human T cells and for T-cell selection

Theodore L Roth; Johnathan Lu; Alison McClellan; Courtney Kernick; Oliver Takacsi-Nagy; Ansuman T Satpathy

doi:10.1038/s41551-025-01372-1

Non-viral intron knock-ins for targeted gene integration into human T cells and for T-cell selection

Nat Biomed Eng. 2025 Mar 7. doi: 10.1038/s41551-025-01372-1. Online ahead of print.

Authors

Theodore L Roth^{1

2

3

4}, Johnathan Lu^{5

6

7

8

9}, Alison McClellan^{5

6

7

8

9}, Courtney Kernick^{5

6

7

8

9}, Oliver Takacsi-Nagy^{5

6

7

8

9}, Ansuman T Satpathy^{10

11

12

13

14}

Affiliations

¹ Department of Pathology, Stanford University, Stanford, CA, USA. troth@stanford.edu.
² Program in Immunology, Stanford University, Stanford, CA, USA. troth@stanford.edu.
³ Stanford Cancer Institute, Stanford University, Stanford, CA, USA. troth@stanford.edu.
⁴ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. troth@stanford.edu.
⁵ Department of Pathology, Stanford University, Stanford, CA, USA.
⁶ Program in Immunology, Stanford University, Stanford, CA, USA.
⁷ Stanford Cancer Institute, Stanford University, Stanford, CA, USA.
⁸ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA.
⁹ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA.
¹⁰ Department of Pathology, Stanford University, Stanford, CA, USA. satpathy@stanford.edu.
¹¹ Program in Immunology, Stanford University, Stanford, CA, USA. satpathy@stanford.edu.
¹² Stanford Cancer Institute, Stanford University, Stanford, CA, USA. satpathy@stanford.edu.
¹³ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA. satpathy@stanford.edu.
¹⁴ Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA. satpathy@stanford.edu.

PMID: 40055580
DOI: 10.1038/s41551-025-01372-1

Abstract

Current methods for the precise integration of DNA sequences into the genome of human T cells predominantly target exonic regions, which limits the choice of integration site and requires complex cell-selection strategies. Here we show that non-viral intron knock-ins for incorporating synthetic exons into endogenous introns enable efficient gene targeting and selective gene knockout in successfully edited cells. In primary human T cells, the knock-in of a chimaeric antigen receptor (CAR) into the T-cell receptor alpha constant locus facilitated the purification of more than 90% CAR⁺ T cells via the negative selection of T-cell-receptor-negative cells. The method is scalable, applicable across intronic sites, as we show for introns within four distinct endogenous surface-receptor genes, and supports the integration of large synthetic exons (longer than 5 kb), of alternative splicing architectures that preserve endogenous gene expression, and of synthetic promoters allowing for endogenous or user-defined gene regulation. Non-viral intron knock-ins expand the range of targetable genomic sites and provide a simplified and high-throughput strategy for selecting edited primary human T cells.