Effective cellular therapies for solid tumors remain elusive. Advances in gene editing and synthetic biology offer great potential for improving the safety and functionality of engineered cellular medicines, but traditional discovery technologies in human immune cells are time-consuming and low-throughput. To overcome these issues, we developed non-viral genome targeting, a method for large-scale genetic engineering of diverse primary human immune cells overcoming the challenges of traditional viral vectors. We further developed pooled knockin screening, enabling rapid discovery of novel sequences to re-wire human immune cell genomes. Further extensions of pooled knockin sequencing have now enabled a high throughput, modular and highly scalable platform to rapidly associate large pools of T cell genetic modifications with high dimensional single cell phenotypes across diverse disease contexts. The resulting genotype x phenotype maps have nominated unique novel cell therapy targets with improved in vitro and in vivo performance for further clinical development.

Learning Objectives:

1. Summarize how synthetic genes can be integrated specifically at endogenous genes to engineer T cell specificity.

2. Describe some benefits of pooled screening systems.

3. Explain how pooled genetic perturbations can be combined with high dimensional single cell phenotypes.