LAUSR

Genetically-engineered immune cell therapies have proven effective to treat some types of cancer, but most tumors still cannot be cured. Forward genetics with CRISPR loss-of-function screens in T cells have identified some mutations to enhance cellular therapies, however these methods do not yet take full advantage of the power of gain-of-function knock-in targeting to engineer cell functions. We developed a robust new platform to assess the functional effects of pools of knock-in constructs in parallel. We used high-efficiency nonviral gene editing to introduce large panels of candidate therapeutic knock-in constructs into a defined genome position in human T cells allowing us to compete the cells against each other and test which constructs enhance T cell function. High-throughput pooled screening of targeted cells identified distinct library members that promoted T cell fitness under various resting, stimulated and immunosuppressive in vitro conditions. Direct competition among adoptively transferred human T cells in immunodeficient mice revealed a subset of constructs that promoted in vivo accumulation of tumor infiltrating lymphocytes. Pooled knock-ins combined with single-cell sequencing also revealed high-dimensional cellular phenotypes induced by each construct ex vivo and in an in vivo tumor microenvironment. Overall, these studies demonstrate the power of pooled knock-in technology to discover and functionally characterize complex synthetic gene programs that can be written into targeted genome sites to generate more effective cellular therapies. This abstract is also being presented as PO073. Citation Format: Theodore L. Roth, Alexander Marson. Highly parallel knock-in targeting for genome engineering of cellular therapies [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR002.