Related Experiment Video
Updated: Jul 16, 2025
11:32
Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines
Published on: November 13, 2021
10.6K
Modular pooled discovery of synthetic knockin sequences to program durable cell therapies
Franziska Blaeschke1, Yan Yi Chen1, Ryan Apathy1
1Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
Cell
|September 15, 2023
Summary
A new screening platform, ModPoKI, enables rapid comparison of genetic modifications to improve T-cell therapies. It identified a transcription factor construct (TFAP4) that enhances CAR-T cell function against cancer.
Area of Science:
- Immunology
- Molecular Biology
- Bioengineering
Background:
- Chronic stimulation impairs T-cell function, limiting cellular immunotherapy efficacy.
- Developing methods to screen numerous genetic modifications is crucial for reprogramming T-cell functions.
- Existing methods lack the scalability to compare large libraries of synthetic sequences.
Purpose of the Study:
- To develop an adaptable platform for high-throughput screening of DNA knockin (KI) libraries.
- To identify genetic constructs that enhance T-cell fitness and anti-cancer function under chronic stimulation.
- To enable the discovery of complex gene combinations for cellular programming.
Main Methods:
- Developed modular pooled KI screening (ModPoKI), a platform for modular DNA KI library construction using barcoded adaptors.
- Created ModPoKI libraries containing 100 transcription factors (TFs) and 129 surface receptors (SRs).
- Performed over 30 ModPoKI screens on human T-cell receptor (TCR)- and chimeric antigen receptor (CAR)-T cells.
Main Results:
- Identified a transcription factor AP4 (TFAP4) construct that significantly enhanced CAR-T cell fitness and anti-cancer activity in vitro and in vivo.
- Demonstrated ModPoKI's modularity by generating a library of approximately 10,000 TF combinations.
- Showcased enhanced T-cell fitness using non-viral KI of a BATF-TFAP4 polycistronic construct, with co-overexpressed BATF and TFAP4 regulating key gene targets.
Conclusions:
- ModPoKI is an effective platform for discovering complex genetic constructs to program cellular functions.
- TFAP4 and BATF co-expression can reprogram T-cell function, enhancing fitness during chronic stimulation.
- This platform accelerates the development of improved cellular immunotherapies.
Keywords:
CRISPRchimeric antigen receptorchronic stimulationhuman T cellsimmunotherapyknockinspooled screenssynthetic surface receptortranscription factor
