In vivo pooled screening for cell therapies: methods development and application to identify genetic drivers of tissue accumulation phenotypes

Abstract

Cell therapy is a transformative approach to treating disease, yet engineering these treatments is challenging due to the complexity of cell behavior across diverse disease contexts. A key limitation is actively delivering therapeutic cells to target tissues, a process dependent on cellular homing, which is universally crucial for the safety and efficacy of cell-based therapies. Overcoming homing deficits has broad implications but is limited by an ability to identify tissue specific effector genes and imposes a requirement for improved in vivo discovery systems. To address this, we introduce two new molecular tools: 1) High-N Barcoding (“HNB”), and 2) Selective Molecular Barcode Enrichment (“SMBE”). Together, HNB and SMBE facilitate genetic library creation and barcode processing, improving construction, resolution, sensitivity, reproducibility, throughput, and cost parameters of in vivo pooled screens. Using these methods, we present the first demonstrations of unbiased, gain-of-function, pooled in vivo screens that enhance CD4 murine T-cell homing to solid tumors. CXCR3 emerged as a significant enrichment across both B16F10-Ova and CT26 tumor models. Validation experiments confirmed that CXCR3 overexpression drives increased T-cell accumulation independent of TCR engagement, suggesting a mechanism centered on CXCR3's role in enhanced T-cell tumor homing relative to GFP controls. Despite CXCR3's known role in mediating T-cell homing to B16 tumors, our study represents the first demonstration that overexpression of CXCR3 can independently drive T-cell accumulation in both B16 and CT26 models. This suggests CXCR3 may be a generalizable strategy to enhance T-cell solid tumor homing. Lastly, we applied our methods to screen a 1500-member "Surface-Ome" library in iPSC- derived myogenic progenitor cells (“MPCs”) in the context of BaCl2-induced injury. This screen identified known and novel genes that enhance MPC engraftment after intramuscular injection, and represents, to our knowledge, the largest in vivo pooled screen using an ORF-based library to study mammalian cell behavior. Taken together, our methods and findings broaden the in vivo functional genomics toolkit and help to advance strategies to engineer and control tissue specific homing for cell-based therapeutic agents.