AND-gated Synthetic Biomarkers for Early Detection of Liver Metastasis

项目摘要 合成生物学的进步将在塑造癌症诊断的未来方面发挥重要作用， 更早、更具体地发现疾病。例如，全细胞生物传感器，如细菌， 基因工程，以执行复杂的功能，如信号放大，以检测临床相关 人尿液和血清中的生物标志物。在哺乳动物细胞中， 布尔逻辑已经被证明用于工程化T细胞疗法的多重控制，从而增加了 肿瘤传感的特异性和降低全身毒性。这些进展突出了合成的前景 然而，这些策略中的绝大多数依赖于遗传电路，并利用 非哺乳动物蛋白质组分。这样的电路是复杂的，并提出了安全性和免疫原性的问题 特别是在体内早期癌症检测的情况下， 生物传感器可能需要监测新生疾病。该提案旨在开发一种新的 诊断称为AND门控合成生物标志物，用于早期检测癌症转移。合成 生物标志物是一类新兴的可激活生物传感器， 系统地，查询早期疾病位点，并利用肿瘤依赖性激活机制，例如 蛋白酶活性失调，以驱动报告基因的产生。这些报告者可以被检测到 非侵入性地从血液、尿液或其他体液样品中分离。蛋白酶在生物学中起着关键的作用。 是转移的主要标志，并且是通过催化不可逆转移而特别有效的分子放大器。 水解肽键，允许单个蛋白酶周转数千个底物。与门选通合成 生物标志物将用于结直肠癌（CRC）肝转移的早期检测。虽然肝脏是一个 原发性CRC转移性扩散的常见部位，肝脏孤立转移瘤的区域切除可能导致 潜在的治疗效果。然而，早期发现CRC肝转移的大小时，他们是最 对治疗有反应的（1-2 mm）仍然具有挑战性的放射成像，如CT和FDA批准的 血液检查，如癌胚抗原（CEA）测试。设计用于CRC的AND门控合成生物标志物 在肝转移中，将基于肝转移中的差异RNA表达来选择蛋白酶的成对组合。 与健康肝组织相比，CRC肝转移。将开发多变量数学模型， 理解设计参数如何与实验结果相比增强特异性和协同性。 CRC肝转移的同基因和异种移植模型将用于临床前验证研究， 以CT和CEA为基准。这一建议将为癌症的早期发现奠定基础 通过可编程的合成生物标志物转移。

PROJECT SUMMARY Advances in synthetic biology will play a fundamental role in shaping the future of cancer diagnostics toward earlier and more specific detection of disease. For example, whole-cell biosensors such as bacteria have been genetically engineered to perform complex functions such as signal amplification to detect clinically relevant biomarkers in human urine and serum. In mammalian cells, sense-and-respond components that employ Boolean logic have been demonstrated for multiplexed control of engineered T cell therapies, thereby increasing the specificity of tumor sensing and reducing systemic toxicity. These advances highlight the promise of synthetic biology when applied to cancer, yet the vast majority of these strategies rely on genetic circuits and make use of non-mammalian protein components. Such circuits are complex and raise safety and immunogenicity concerns for regulatory approval, especially in the context of in vivo early cancer detection where repeated administrations of biosensors are likely needed to monitor for nascent disease. This proposal seeks to develop a new class of diagnostics called AND-gated synthetic biomarkers for early detection of cancer metastasis. Synthetic biomarkers are an emerging class of activatable biological sensors that are designed to be administered systemically, query sites of early disease, and harness tumor-dependent activation mechanisms, such as dysregulated protease activity, to drive production of a reporter. These reporters can then be detected noninvasively from blood, urine, or other bodily fluid samples. Proteases play key biological roles across the major hallmarks of metastasis and are particularly potent molecular amplifiers by catalyzing the irreversible hydrolysis of peptide bonds, allowing a single protease to turnover thousands of substrates. AND-gated synthetic biomarkers will be applied for early detection of colorectal cancer (CRC) liver metastasis. Although the liver is a common site for metastatic spread from primary CRC, regional resection of liver-isolated metastases can lead to potentially curative results. Yet early detection of CRC liver metastases at a size when they are most responsive to therapy (1–2 mm) remains challenging by radiographic imaging such as CT and FDA-approved blood test such as the carcinoembryonic antigen (CEA) test. To design AND-gated synthetic biomarkers for CRC liver metastasis, pairwise combinations of proteases will be selected based on differential RNA expression in CRC liver metastases compared to healthy liver tissue. Multivariate mathematical models will be developed to understand how design parameters enhance specificity and cooperativity compared to experimental results. Syngeneic and xenograft models of CRC liver metastasis will be used for preclinical validation studies to allow benchmarking against CT and CEA. This proposal will lay the groundwork for earlier detection of cancer metastasis by programmable synthetic biomarkers.