P53 and KRAS Targeted Pigs: A Platform for Models of Human Cancer

项目总结/摘要 癌症是美国第二大致命疾病，每年导致50多万美国人死亡。今年，另有150万人将被诊断出患有近200种不同癌症类型之一。尽管对环境危险因素、遗传因素和肿瘤发生机制的认识不断加深，但对该病的诊断和治疗仍然不足。关于癌症的大部分知识都来自于对培养细胞和小动物模型的研究。虽然这些模型系统提供了非常丰富的信息，但它们也具有局限性，并且在将有前途的疗法转化为临床方面存在挑战。缺乏准确复制人类癌症的大型动物模型一直是开发这种致命疾病的有效诊断工具，干预措施和疗法的主要障碍。猪在解剖学、生理学、遗传学以及重要的体型方面与人类有许多相似之处。虽然由于标准的猪肉生产实践，自然发生的肿瘤很少见到，但自发性和诱导性癌症已在猪中进行了研究，并且高度代表了在人类中观察到的情况。然而，在猪中诱导癌症所需的延长的时间范围和伴随的表型变异性限制了它们的有用性。我们的目标是创造一种基因工程猪，其中肿瘤发生可以在任何组织中有条件地诱导。我们打算通过突变人类癌症中最常见的两个基因KRAS和TP 53（编码p53）来实现这一目标。KRAS是一种癌基因，编码一种小的GT3，它与受体激活和下游效应物偶联，控制细胞增殖、分化和存活。KRAS的激活突变在许多人类肿瘤中很常见。p53被称为“基因组的守护者”，是一种转录因子，调节细胞的重要功能，包括细胞周期停滞和凋亡。适当的p53功能的丧失使细胞倾向于不受调节的生长、肿瘤形成和转移。表达KRAS和TP 53条件突变的小鼠模型已经产生了代表人类肺癌、胰腺癌、结肠癌和其他组织癌的优秀模型。我们假设猪KRAS和TP 53的突变将在猪中产生类似的结果。因此，该项目的最终目标是开发和商业化KRAS/TP 53-突变猪，作为人类癌症模型的平台。我们打算通过结合基因打靶和体细胞核移植来实现这一目标。该提案具体概述了具有突变的KRAS和TP 53等位基因的猪成纤维细胞的开发。将开发一种基因靶向载体，并用于在TP 53靶向细胞和野生型细胞中通过同源重组破坏内源性猪KRAS。后续工作将利用这些细胞作为核供体进行体细胞核移植，生产KRAS/TP 53-突变猪。这些模型将为学术和行业研究人员提供更好地了解癌症及其发病机制的机会，并开发和测试新的诊断，治疗和预防策略。

Project Summary/Abstract Cancer is the second deadliest disease in the United States, killing more than 500,000 Americans annually. This year, another 1.5 million will be diagnosed with one of nearly 200 different cancer types. Despite an ever---growing understanding of the environmental risk factors, genetic contributions, and tumorigenic mechanisms, the diagnoses and treatments for this disease remain inadequate. Much of what is known about cancer has come from studies of cells in culture and small animal models. While these model systems have been extremely informative, they also possess limitations and present challenges to translating promising therapies to the clinic. The lack of a large animal model that accurately replicates human cancer has been a major barrier to the development of effective diagnostic tools, interventions, and therapies for this deadly disease. Pigs share many similarities with humans in anatomy, physiology, genetics, and importantly, size. While naturally occurring tumors are rarely seen due to standard pork production practices, spontaneous and induced cancers have been studied in pigs and are highly representative of what is seen in humans. However, the extended timeframe required for inducing cancer in pigs and the accompanying phenotypic variability limit their usefulness. Our objective is to create a genetically engineered pig in which tumorigenesis can be conditionally induced in any tissue. We intend to accomplish this by mutating two of the most commonly affected genes in human cancer, KRAS and TP53 (encoding p53). KRAS is in oncogene encoding a small GTPase that couples receptor activation and downstream effectors to control cell proliferation, differentiation and survival. Activating mutations in KRAS are common in many human tumors. Known as the "guardian of the genome", p53 is a transcription factor that regulates critical cell functions including cell---cycle arrest and apoptosis. The loss of proper p53 function predisposes cells to unregulated growth, tumor formation, and metastasis. Mouse models expressing conditional mutations in KRAS and TP53 have yielded excellent models representative of human cancers of the lung, pancreas, colon, and other tissues. We hypothesize that mutations in porcine KRAS and TP53 will produce similar results in pigs. Therefore, the ultimate goal of this project is to develop and commercialize KRAS/TP53--mutated pigs to serve as a platform for models of human cancer. We intend to accomplish this by combining gene targeting and somatic cell nuclear transfer. This proposal specifically outlines the development of porcine fibroblasts with mutated KRAS and TP53 alleles. A gene targeting vector will be developed and used to disrupt the endogenous porcine KRAS via homologous recombination in both TP53---targeted and wild---type cells. Subsequent work will use these cells as nuclear donors for somatic cell nuclear transfer to produce KRAS/TP53---mutated pigs. These models will provide academic and industry researchers with an opportunity to better understand cancer and its pathogenesis and to develop and test new diagnostic, therapeutic, and preventative strategies.