Jensen, Michael COda, Shannon KCurtis, Benjamin C2023-08-142023-08-142023Curtis_washington_0250E_25469.pdfhttp://hdl.handle.net/1773/50518Thesis (Ph.D.)--University of Washington, 2023Chimeric antigen receptor (CAR) T cell therapy has revolutionized cancer care through genetic reprogramming, and next-generation cell engineering aims to encode increasingly complex genetic programs to further modulate T cell behavior, and ultimately potentiate anti-tumor activity. Effective selection of coding sequences for subsequent clinical development requires one or multiple prioritization regimens, but scalable gene nomination platforms for T cell therapy are currently lacking. In this work, I describe the creation of a novel genome-scale, gain-of-function screening platform, powered by CRISPR activation (CRISPRa) technology, and designed for pooled screening in primary human CAR T cells. To achieve this, established CRISPRa designs were first optimized for fidelity, potency, and durability in T cell lines. Second, restrictive primary T cell requirements were overcome through cell manufacturing featuring transposon-based gene delivery coupled to CAR-restricted expansion (EP-TICLE). To overcome abbreviated CRISPRa activity, CRISPRa expression was reinforced through construct optimization that included dual divergent hybrid promoters (EF1α-HTLV)2 and intronization of the dCas9 coding cassette. Finally, to prevent library distortion, gRNAs were delivered in trans via lentivirus immediately prior to landscape initiation. This manufacturing schema was trialed at genome-scale and nominated modulators of CAR T cell homeostatic persistence (MYC, STAT5A, STAT5B) and cytokine-free survival (CSF2RB, TNFRSF1A, TNFRSF1B). Follow-up studies suggested that this platform would tolerate a druggable-ON switch (ERT2) to regulate landscape initiation. In sum, this work demonstrates the utility of a novel cell manufacturing and gain-of-function screening system for next-generation therapy development. Insights into achieving sustained dCas9 expression may also offer a blueprint for orthogonal CRISPR-based screening systems or for in vivo CRISPR applications.application/pdfen-USnoneCARCRISPRCRISPRaBioengineeringImmunologyPathologyThesis