Engineering Therapeutic Human Immune Cells with Modular Self-contained Genetic Circuits

项目总结/摘要 目前工程化基于人类细胞的治疗剂的策略依赖于递送和随后的治疗。 转基因有效载荷的基因组整合。尽管这些方法已经催化了变革性的 随着医学的进步，转基因DNA的整合永久性地破坏了天然基因组序列 并可能导致意想不到的甚至危险的后果。此外，整合的转基因DNA 通常不可预测地表达，并且随着时间的推移，特别是在原发性肿瘤中， 人体免疫细胞此外，现有的方法来验证大的转基因组- 整合的DNA货物是低效且昂贵的。这些关键的障碍限制了人类 细胞可以被重新利用和改造为基于细胞的治疗方法，这些挑战正在阻止 生物技术和临床创新。非整合的双链DNA病毒已经进化 这些关键障碍的复杂解决方案，它们可以稳定地存在于人体细胞中， 环化的自含游离体跨细胞分裂和感染宿主的寿命。这些 病毒通过调整它们自己的基因表达模式来实现这种非凡的持久性， 同步其基因组复制，并通过重塑内源性转录网络， 宿主细胞在本提案中，我们将利用这些自然能力，并使用临床级 基因治疗载体试验台。我们的方法将建立一种全新的方式来使用循环， 人细胞内的正交附加型DNA。 在本提案的目标1中，我们设计、构建、测试和优化遗传编码的附加体模块， 使i）位点特异性和可调的基因组定位，ii）可编程的附加型复制，和iii） 多层安全开关，在临床验证的整合酶缺陷型慢病毒（IDLV）和高 容量腺病毒（HcAdV）基因治疗载体试验床。在本提案的目标2中，我们将建立遗传 IDLV和HcAdV基因治疗载体内的回路，其感测缺氧环境和/或小的 分子，并通过产生荧光诊断来真实的响应这些信号，和/或 合成的基于CRISPR/Cas9的转录因子，以驱动治疗关键基因的表达， 细胞因子或生物医学相关的表型变化。在每个独立的目标中，我们将使用 功能基因组学、基因组工程和合成生物学的实验技术 生物学为了保持和最大限度地提高我们的结果的治疗效用，并确保适用性超越 在本提案的范围内，两个目标都将使用原代人T细胞和间充质干细胞进行。 基质细胞总的来说，这个项目将结合联合收割机工程原理和生物医学的经验教训， 科学促进进步，这将是广泛有用的生物医学研究人员，可为临床医生， 并且对未来需要复杂的基于细胞的疗法的患者有意义。

PROJECT SUMMARY/ABSTRACT Current strategies to engineer human cell-based therapeutics rely upon the delivery and subsequent genomic integration of transgenic payloads. Although these approaches have catalyzed transformative medical advances, the integration of transgenic DNA permanently disrupts natural genomic sequences and can lead to unexpected and even hazardous consequences. In addition, integrated transgenic DNA is often unpredictably expressed and is prone to epigenetic silencing over time, especially within primary human immune cells. Furthermore, existing approaches to validate large transgenic genomically- integrated DNA cargoes are inefficient and costly. These critical barriers limit the extent to which human cells can be repurposed and engineered as cell-based therapeutics and these challenges are preventing biotechnological and clinical innovations. Non-integrating, double-stranded DNA viruses have evolved sophisticated solutions to these critical barriers, and they can stably persist within human cells as circularized self-contained episomes across cellular divisions and for the lifetime of infected hosts. These viruses accomplish this remarkable persistence by tailoring their own gene expression patterns, synchronizing their genomic replication, and by reshaping the endogenous transcriptional networks of host cells. In this proposal, we will harness these natural abilities and refine them using clinical-grade gene therapy vector testbeds. Our approach will establish an entirely new way to use of circular, orthogonal episomal DNA within human cells. In Aim 1 of this proposal, we design, build, test, and optimize genetically-encoded episomal modules to enable i) site-specific and tunable genomic localization, ii) programmable episomal replication, and iii) multi-layered safety switches, within clinically validated integrase-deficient lentiviral (IDLV) and high- capacity adenoviral (HcAdV) gene therapy vector testbeds. In Aim 2 of this proposal, we will build genetic circuits within IDLV and HcAdV gene therapy vectors that sense hypoxic environments and/or small molecules and respond to these signals in real time by producing fluorometric diagnostics and/or synthetic CRISPR/Cas9-based transcription factors to drive the expression of therapeutically crucial cytokines or biomedically relevant phenotypic changes. In each independent Aim, we will use experimental techniques at the interface of functional genomics, genome engineering, and synthetic biology. To preserve and maximize the therapeutic utility of our results and to ensure applicability beyond the scope of this proposal, both Aims will be carried out using primary human T cells and mesenchymal stromal cells. Collectively, this project will combine engineering principles and lessons from biomedical sciences to spur advances that will be broadly useful to biomedical researchers, actionable for clinicians, and meaningful to future patients in need of sophisticated cell-based therapeutics.