Prime-CAR Inspection: Enhancing Cancer-Directed CAR-T Cells with Safety-Validated CRISPR Prime Editors

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In Prime-CAR Inspection, I will unleash the potential of CRISPR prime editing (PE), a novel and transformative CRISPR gene editing platform with unprecedented versatility, to precision-engineer cancer-directed CAR-T cells. PE can install all single nucleotide changes, small deletions, and insertions without inducing DNA double-strand breaks (DSB) or requiring a DNA template. PE is ideal to precision-edit CAR-T cells without risking undesired consequences of DNA DSBs associated with conventional CRISPR/Cas9 gene editing. However, to my knowledge, PE-engineered CAR-T cell products do not yet exist due to PE's recent development. PE expertise in Germany is currently limited. The Emmy Noether Program is excellent for attaining scientific independence. I aim to successfully establish an Emmy Noether group and apply advanced PE concepts and state-of-the-art molecular methods to generate safety-validated PE-engineered CAR-T products (Prime-CAR-Ts). To do this, I will (1) rapidly develop Prime-CAR-T prototypes installing DSB-free hypomorphic and PE-exclusive TGF-ß and PD-1 variants, validate PE's genomic safety with oligonucleotide enrichment and sequencing (ONE-seq), a molecular method I developed in my postdoc, and in-depth analyze Prime-CAR-T products in vitro and in vivo. Next, I will (2) profile targeted CAR transfer using PE-guided recombination, validate genomic safety with integration site analysis (extensively applied in my doctoral work), and systematically evaluate CAR-T products in cell and mouse models. Finally, I will (3) high-throughput profile PE in primary human T cells at more than 10,000 immune-relevant sites in parallel using library approaches and next-generation sequencing, accelerating PE's adaptation to primary T cells and potentially other CAR-targetable cells. The key ambition of Prime-CAR Inspection is to materialize PE's incredible potential for CAR-T therapy and to pave the way for its clinical translation. The suggested work program is strategically structured and ambitiously designed to ensure rapid output and high impact to the benefit of patients and the scientific community, but it also provides a trajectory to an independent, productive, and sustainable research program. I will continue developing PE and safety validation technology in Germany. I selected Würzburg as the research site because I felt inspired by the impressive work of the translational immunotherapy program at the Universitätsklinikum Würzburg, which is highly synergistic with my research ambitions. I assessed the Würzburg research environment and found it to be maximally supportive and optimally suited to conduct Prime-CAR Inspection and follow-up research. I bring a network of international collaborators and have already started to form a network with collaborators in Würzburg. Prime-CAR Inspection will enable the adaptation of PE to CAR-T cell engineering and therapy with the goal of improving clinical care and outcomes for cancer patients.