A Workflow for In Vivo Evaluation of Candidate Inputs and Outputs for Cell Classifier Gene Circuits

Cell classifier gene circuits that integrate multiple molecular inputs to restrict the expression of therapeutic outputs to cancer cells have the potential to result in efficacious and safe cancer therapies. Preclinical translation of the hitherto developments requires creating the conditions where the animal model, the delivery platform, in vivo expression levels of the inputs, and the efficacy of the output, all come together to enable detailed evaluation of the fully assembled circuits. Here we show an integrated workflow that addresses these issues and builds the framework for preclinical classifier studies using the design framework of microRNA (miRNA, miR)-based classifier gene circuits. Specifically, we employ HCT-116 colorectal cancer cell xenograft in an experimental mouse metastatic liver tumor model together with Adeno-associated virus (AAV) vector delivery platform. Novel engineered AAV-based constructs are used to validate in vivo the candidate inputs miR-122 and miR-7 and, separately, the cytotoxic output HSV-TK/ganciclovir. We show that while the data are largely consistent with expectations, crucial insights are gained that could not have been obtained in vitro. The results highlight the importance of detailed stepwise interrogation of the experimental parameters as a necessary step toward clinical translation of synthetic gene circuits.可整合多种分子输入信号、仅在癌细胞中特异性启动治疗性输出表达的细胞分类器基因回路，有望催生高效且安全的癌症治疗策略。针对目前已取得的相关研究进展，要实现其临床前转化，需要构建适配的实验体系，使动物模型、递送平台、输入分子的体内表达水平以及输出分子的治疗疗效得以协同配合，从而对完整组装的基因回路开展全面详尽的评估。本研究提出一套整合式工作流程，以基于微小RNA（microRNA，miRNA，miR）的分类器基因回路设计框架为基础，搭建了临床前分类器研究的标准化体系。具体而言，我们采用HCT-116结直肠癌异种移植瘤构建实验性小鼠转移性肝肿瘤模型，并联合腺相关病毒（Adeno-associated virus，AAV）载体递送平台。我们借助全新工程化改造的AAV载体构建物，在体内分别验证了候选输入分子miR-122、miR-7，以及细胞毒性输出元件HSV-TK/更昔洛韦（ganciclovir）。研究结果表明，尽管整体数据与预期相符，但我们仍获得了体外实验无法获取的关键认知。本研究结果凸显了对实验参数进行细致逐步骤探究的必要性，这是合成基因回路迈向临床转化的必要前置步骤。