Mutant p53 mouse cancer models using somatic CRISPR base editing

TP 53编码肿瘤抑制转录因子p53，是癌细胞中最常突变的基因。与其他肿瘤抑制基因不同，TP 53最常受到错义突变的打击，导致肿瘤抑制活性（可变）降低。此外，突变型p53蛋白的积累可能导致显性负性和新形态（功能获得）活性，促进肿瘤进展并构成潜在的治疗靶点。然而，已经描述了2000多个功能多样的错义突变，因此需要深入了解单个突变体及其后果，以便更好地利用TP 53状态进行个性化治疗决策。基因工程p53突变敲入小鼠已成为有价值的临床前工具，并大大推进了我们对突变p53在肿瘤发生和癌症治疗中的功能的理解。然而，与生殖系基因敲入相关的潜在努力和成本阻碍了对多种突变的系统性体内比较和对p53突变功能多样性的更深入了解。在本项目中，我们计划利用体细胞基因组编辑的最新进展，使用先进的CRISPR工具来比较体内不同的p53突变。大多数具有p53错义突变的肿瘤是由C至T转换引起的，其可以用胞嘧啶碱基编辑器（CBE）工程化，即由Cas9切口酶、胞嘧啶脱氨酶和尿嘧啶-N-糖苷酶抑制剂组成的融合蛋白。在我们的初步工作中，我们已经开发了一种具有上级碱基编辑效率的改进的CBE，其允许精确诱导用先前描述的CBE不能产生的各种p53点突变。此外，我们显示了使用递送经典CRISPR-Cas9核酸酶的腺病毒载体对成年小鼠的气管内感染成功地体细胞诱导小细胞肺癌（SCLC）。我们现在计划将这些技术联合收割机结合起来，并使用腺病毒递送CBE来产生在p53和协作驱动基因中具有确定的单核苷酸取代（错义和无义突变）的SCLC肿瘤。将全面系统地分析所得肿瘤细胞和肿瘤微环境表型。这些研究有望为小细胞肺癌定义的体内背景下与不同p53突变相关的功能多样性提供深入和前所未有的见解。

TP53 encodes the tumor suppressive transcription factor p53 and is the most frequently mutated gene in cancer cells. Different to other tumor suppressor genes, TP53 is most commonly hit by missense mutations which lead to a (variable) reduction in tumor suppressor activity. In addition, accumulation of mutant p53 proteins may result in dominant-negative and neomorphic (gain-of-function) activities that promote tumor progression and constitute a potential therapeutic target. However, more than 2000 functionally diverse missense mutations have been described, so that an in-depth understanding of single mutants and their consequences is needed to make better use of the TP53 status for personalized treatment decisions. Genetically-engineered p53 mutant knock-in mice have become valuable preclinical tools and have massively advanced our understanding of mutant p53 functions in tumorigenesis and cancer therapy. However, the underlying effort and costs associated with generating germline knock-ins preclude a systematic in vivo comparison of multiple mutations and deeper insight into the functional diversity of p53 mutations.In this project, we plan to make use of recent progress in somatic genome editing with advanced CRISPR tools to compare different p53 mutations in vivo. Most tumors with a p53 missense mutation are caused by C-to-T transitions which can be engineered with cytosine base editors (CBE), i.e. fusion proteins consisting of a Cas9 nickase, a cytosine deaminase and a uracil-N-glycosidase inhibitor. In our preliminary work, we have developed an improved CBE with superior base editing efficiency that allows the precise induction of various p53 point mutations which could not be generated with previously described CBEs. Moreover, we show successful somatic induction of small cell lung cancer (SCLC) using intratracheal infection of adult mice with adenoviral vectors delivering classical CRISPR-Cas9 nucleases. We now plan to combine these technologies and use adenoviral delivery of CBEs to generate SCLC tumors with defined single nucleotide substitutions (missense and nonsense mutations) in p53 and cooperating driver genes. The resulting tumor cell and tumor microenvironmental phenotypes will be profiled comprehensively and systematically. These studies are expected to provide deep and unprecedented insight into the functional diversity associated with distinct p53 mutations in the defined in vivo context of small cell lung cancer.