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

在Prime-Car检查中，我将释放CRISPR Prime Editing(PE)的潜力，这是一个具有前所未有的多功能性的新型变革性CRISPR基因编辑平台，用于精确设计癌症导向的CAR-T细胞。PE可以安装所有单核苷酸改变、小的删除和插入，而不会导致DNA双链断裂(DSB)或需要DNA模板。PE是精确编辑CAR-T细胞的理想选择，而不会冒与传统CRISPR/Cas9基因编辑相关的DNADSB的不良后果。然而，据我所知，由于PE最近的发展，PE工程的CAR-T细胞产品还不存在。德国的体育专业知识目前有限。艾美诺特计划在实现科学独立方面非常出色。我的目标是成功地建立一个艾美诺特小组，并应用先进的PE概念和最先进的分子方法来生产安全验证的PE工程CAR-T产品(Prime-CAR-TS)。为此，我将(1)快速开发安装不含DSB的亚晶型和PE专有的转化生长因子和PD-1变异体的Prime-CAR-T原型，使用我在博士后开发的分子方法-寡核苷酸浓缩和测序(One-Seq)验证PE的基因组安全性，并深入分析Prime-CAR-T产品的体外和体内。接下来，我将(2)介绍使用PE引导的重组的靶向CAR转移，通过整合位点分析验证基因组安全性(在我的博士工作中广泛应用)，并在细胞和小鼠模型中系统地评估CAR-T产品。最后，我将(3)使用文库方法和下一代测序方法，并行地在10,000多个免疫相关部位的原代人类T细胞中高通量分析PE，加速PE对原始T细胞和潜在的其他靶向细胞的适应。Prime-Car检查的主要目标是实现PE在CAR-T治疗方面令人难以置信的潜力，并为其临床翻译铺平道路。建议的工作计划具有战略性的结构和雄心勃勃的设计，以确保快速产出和对患者和科学界的好处，但它也提供了一个独立、多产和可持续的研究计划的轨迹。我将继续在德国开发PE和安全验证技术。我选择维尔茨堡作为研究地点，是因为我受到维尔茨堡大学转化免疫疗法项目令人印象深刻的工作的启发，这与我的研究雄心高度协同。我评估了维尔茨堡的研究环境，发现它最大限度地支持和最适合进行优质汽车检查和后续研究。我带来了一个国际合作者网络，并已经开始与维尔茨堡的合作者建立网络。Prime-CAR检查将使PE适应CAR-T细胞工程和治疗，目标是改善癌症患者的临床护理和预后。

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.